MTI

Question 1

What was the first form of genetic material

Answer 1

RNA was the first form of genetic material, developed into DNA for storage without degradation because it is more stable, and protein for function-more efficient and versatile than RNA, evolution and phenotype

Question 2

What links genotype and phenotype

Answer 2

RNA

Question 3

Name some functions of RNA

Answer 3

Store information
Mediate catalysis
RNA first used to store info and mediate catalysis (called ribozymes)

Question 4

What determined the chemical building blocks of life

Answer 4

Prebiotic Synthesis determined the chemical building blocks of life (amino acids, nucleotides, sugars and fatty acids)

Question 5

Self replicating systems allow what

Answer 5

Self replicating systems allowed natural selection to operate

Question 6

What are some reasons DNA overtook RNA

Answer 6

• DNA is more efficient than RNA
• Less susceptible to degradation and hydrolysis
• DNA stores genotypic info while RNA functions as the intermediate leading to protein synthesis (RNA still also functions through catalysis, structure, and phenotype

Question 7

Describe the original cells (first true cells)

Answer 7

• Original cells were VERY simple; even modern day mycoplasma are much more complex than these early entities
  NOTE-all cells have evolved for the same length of time

Question 8

True/False: All cells have evolved for the same length of time

Answer 8

True!

Question 9

How are prokaryotes and eukaryotes defined

Answer 9

By the presence of a nucleus

Question 10

Eukaryote

Answer 10

· Eukaryotes-strucutrally complex
§ Definded by presence of nucleus
§ Evolved to replicate genome and efficiently transcribe DNA into RNA
§ Double layer nuclear envelope
§ Contains internal membrane structures
§ Follows volume to surface area rules for size of limitations/growth
§ Ex: large eukaryotic cells have more volume than surface area
· Mitochondira-most eukaryotes have mitochondria in cytoplasm
o Double membrane compartments specialized for oxidative phosphorylation and energy production
o Thought to have come from endosymbiosis
§ Consistent with mitochondria having their own DNA genomes and primitive transcriptional/translational machinery
· Lysosomes-contain hydrolytic enzymes involved in turn over and degradation of cell components
· Peroxisomes-oxidative enzymes that generate and destroy hydrogen peroxide; involved in lipid metabolism
· Chloroplasts-in addition to having mitochondria and are specialized for photosynthesis

Question 11

What are the various model organisms? Why do we use them?

Answer 11

· The various model organisms-worms, flies, mice, human and Arabidopsis
· We use model organisms because they have high birth and death rates and we can compare these results to humans. These are organisms with eukaryotic cells; so results from these tests do not stray too far from that which is true of humans

Question 12

Light Microscope

Answer 12

Light microscope (LM): requires solid state lens, not power Limitation: lower resolution than EM can’t visualize live sample/require fixing

Question 13

Solution to limitation

Answer 13

Solution to limitation: confoceal scanning and deconvolution microscopes restrieting excitation of fluorescent image to a single plain within a cell then use computational method to process and put images together

Question 14

Electron Microscope

Answer 14

Electron microscope (EM): have better resolution (100x). use x rays and gamma rays based on dual wave/particle nature to matter. Method relies on accelerating electron diffraction similar to that of protons, then focus on accelerated electrons using magnet.

Question 15

The general requirements for propagation of cells in culture

Answer 15

The general requirements for propagation of cells in culture
· First requirement is that the cells must able to be cultured. Most bacteria we are aware of will not grow in lab environments and we cannot study using cultures

Question 16

The principles behind subcellular and macromolecular fractionation including gradient centrifugation, chromatography

Answer 16

The basis behind methods of tracing olecules inside cells

Question 17

The general structure of lipids and lipid bilayers

Answer 17

o The lipid bilayer is the universal basis for cell membrane structure
o Composed of lipids
§ Amphipathic molecules-insoluble in water, highly soluble in organic solvents, have a polar end composed of phosphate and sugars and a hydrophobic end composed of fatty acids
§ Their role may be protective to affect the electrical properties of the bilayer or they may be involved as recognition molecules in cell to cell interactions
· Bilayer behaves as 2 dimensional fluid
o Lipids in the bilayer can diffuse rapidly in the plane of the membrane circumnavigate the surface
o Lipids must be aided to flip from one surface to the other-phospholipid translocators are proteins that assist in this movement
· Membrane fluidity and factors that influence it
o Temperature
§ High temp-more fluid
§ Low emp-stiffens
o Chemical composition
o Longer fatty acid chain more saturated less fluid bilayer
o More cholestrerol depends on which layer it is added to but in general more impermeable
Means by which proteins associate with membranes
Peripheral membrane proteins
Associate with the membrane through noncovalent interactions with other

Question 18

Integral membrane proteins

Answer 18

Held in the bilayer of lipid groups or by hydrophobic interactions inserted directly into the lipid bilayer
Delivered to the membrane via transport vesicles
Folding in a way that embeds hydrophobic regions within a monolayer or bilayer
Covalently bonding to a fatty acid chain embedded in the monolayer
Having one whole protein in the aqueous region and anchored to the membrane
In addition to being a transmembrane protein to increase its own hydrophobicity
Through another covalent linkage to a oligosaccharide
Protein mobility in lipid bilayers and methods by which lipids and proteins can be confined to specific domains of membranes
- Proteins associated with only one layer of the bilayer
- Carry out functions on that same side of the bilayer
- Can be either integral or peripheral, can be directly or indirectly associated with the bilayer

Question 19

Transmembrane proteins

Answer 19

Transmembrane proteins span across layer and bilayer
Carry out functions of both sides of bilayer
Allow passage for hydrophilibic molecules through hydrophobic bilayer

Question 20

What does active transport require

Answer 20

ATP (some kind of energy)

Question 21

Ion Channels

Answer 21

Ion channels form “resting membrane potential” to allow the transport of ions through themselves. They are activated by different triggers such as voltage, ligands, and mechanic actions. Voltage gated channels is open when its favored by the charges on the membrane

Question 22

Ligand-gated channels

Answer 22

Ligand-gated channels are opened when the come in contatct with some ligand. Mechanically-gated channels physically push themselves and their way aprt to open

Question 23

Ion Pumps

Answer 23

Ion pumps require energy to work common example is a sodium potassium pump that uses ATP and pumpes out Na+ and creates an electrochemical gradient

Question 24

Symporter

Answer 24

Symporter is a transporter that is coupled and transports material in or out two at a time. Symmetrical porter

Question 25

Antiporter

Answer 25

Antiporter is a coupled transporter that transports material in and out simultaneously. Anti=against

Question 26

Mitochondria-TOM Complex

Answer 26

Mitochondria-TOM complex is on the outer membrane of mitochondria and TIM complexes are on the inner membrane of mitochondria to facilitate the movement of proteins inside. Regular proteins just through both and end up inside in the mitochondrial matrix. Some proteins have a second signal so once they’re in the matrix they’ll go back up through the TIM complex and become intermembrane-space proteins. And those proteins that are destined for membrane surfaces are mediated by SAM complexes which are found in the outer membrane and allow correct folding to make integral surface proteins

Question 27

Chloroplasts

Answer 27

Chloroplasts: much like mitochondria, translocator proteins are needed to transport proteins through the outer membrane and the inner membrane. Chloroplasts have another membrane within through. There are 4 mechanisms by which proteins may enter the thylakoid (within the stroma of the chloroplast). There is the sec pathway requiring ATP, the SRP pathway that requires ATP, the ph pathway requiring some proton gradient, and spontaneous insertion which requires no external energy

Question 28

Membrane potential generated mainly by

Answer 28

Membrane potential generated mainly by Na+ K+
3 Na+ out 2 K+ in
Makes outside more and inside less
Also K leak channels contribute (membranes more permeable to K)
Equilibrium reached as K leaks out, voltage develops and drives K back in (Nerst Equation)

Question 29

Voltage gated K+ channel

Answer 29

Voltage gated K+ channel
4 identical subunits
B-sheet determines permeability characteristics
N terminal domain on cytoplasmic side as plug
Series of amino acids in one of the domains senses voltage gradient and opens channel when voltage falls

Question 30

the nature of actions of voltage gated channels, and how they generate and sustain action potentials in nerve and muscle cells

Answer 30

Voltage gated Na channels open when voltage across membrane decreases
Causes Na channels open in adjacent patches
Then causes adjacent voltage drops
Wave of depolarization down axon
Na channels unstable when open
Close quickly, K leaks restore resting membrane potential (negative voltage)
Voltage gated delayed K channels can open when membrane potential begins to drop and help further restore
Peripheral/CNS nerves-surrounded by mycelin
Synapses can be stimulatory or inhibitory

Question 31

transmission of nerve impulses across synapses, particular at the neuromuscular junction

Answer 31

Neuromuscular junction-where terminus of axon spreads over specialized regions of muscle cell
Axon terminus contains secretory vesciles with acetylcholine (neural transmitter)
Acetylchloline receptor on the nerve cell binds acetylcholine, changes confiramation of subunits and opens channel letting na into muscle cell
Depolarization of muscle cell triggers voltage gated Na channels
Propagates depolarization across entire surface of muscle cell
Depolarization also causes Ca channels in muscle cell to open
Release Ca from sarcoplasmic reticulum
Free Ca in cytoplasm of muscle cell induces actin/myosin complex to contract
Synaptic transmission stopped with acetylcholinesterase
Destroys neurotransmitter

Question 32

Effects of drugs

Answer 32

Prevents synapse from resetting-muscle depolarized and contracted
Used in insecticides

Question 33

Bungarotoxin

Answer 33

Prevents acetyl choline receptor channel from opening

Found in some snake venoms

Question 34

Curare

Answer 34

Blocks the channel

Used in surgery

Question 35

General principles of cell signaling

Answer 35

Mostly transmembrane signals-signals from one cell to another cell crossing a membrane
Types of cell-cell signaling by secreting molecules
Contact dependent-signaling cell with membrane bound signaling molecule comes close to its target cell binds the molecule to the membrane bound receptor
Paracrine-signaling cell sends signals to nearby cells, works locally
Synaptic-signaling is close by, transmitted by small chemicals within a synapse
Signal works locally even if the origin is far away
Specificity is determined by the proximity of the target cells and receptors expressed
Endocrine-signals originate from endocrine cell which transports signal molecules through the bloodstream to the faraway target cell
Specificity determined by receptors expressed on target celss, not proximity
Autocrine-signaling cell releases signal molecules for its own receptors
Form of quorum sensing (multicellular slimemolds)

Question 36

The general structure and nature of G protein-linked receptors and their mechanism of action through trimerie G proteins

Answer 36

The two major downstream intracellular signals mediated through G protein coupled receptors cAMP and Ca++
The effects of cAMP and Ca++ on protein kinases and the roles of calmodulin and troponin D

Question 37

How did systems become more efficient (evolutionarily)

Answer 37

Proteins are more versatile catalysts than RNA (20 possible flavors of amino acid, plus modifications, compared to only 4 types of bases in RNA), therefore the development of translation, allowing a sequence of RNA to be converted to a protein, was a major step in evolution of phenotype (in fact, this evolution has been continuing, and there is evidence that certain amino acids have been incorporated more recently into the genetic code than have others). Similarly, DNA is a more stable form of genetic material than RNA (a major improvement for archival storage of genotype- DNA is significantly more resistant to degradation and to hydrolysis than is RNA). Most life as we now see it uses DNA as genotype and RNA as intermediate leading to protein synthesis, catalysis, structure, and phenotype.

Question 38

What memorie of RNA still exist today

Answer 38

viruses that use RNA as genome (influenza, HIV), catalytic RNAs at the heart of ribosome function and mRNA splicing. Ironically, in fact, the actual catalytic processes involved in the translation of proteins in ribosomes is virtually all RNA based, with the ribosomal proteins added just to improve efficiency and stability.

Question 39

Evolution of the first true cells

Answer 39

Early life probably evolved in complex organic soup, yet ultimately needed to create barriers, to separate biological reactions (dependent on defined conditions) from the more variable external environment. Also-no selective advantage to a particular RNA able to encode an enzyme if the enzyme diffuses and operates elsewhere in soup. First true cells were defined by creation of first cell membranes- composed of lipids.

Question 40

Mycoplasma

Answer 40

Even simplest of present day cells are much more complex than these early entities at border of life and non-life. As example- modern day mycoplasma. Small (0.3 um) bacteria-like organisms with cell membranes, but no cell walls. Mycoplasmal genome is enough to encode about 482 proteins (enzymes, structural). Amazingly, this is sufficient to encode all enzymes necessary for metabolism, generation and storage of chemical energy, DNA replication, repair, transcription, and translation, and well as all the processes necessary to accumulate or synthesize the required biochemical precursors and all the structural proteins needed to maintain the cell shape and integrity. In fact, investigators have recently created replaced the natural genome in Mycoplasma with a completely synthesized one, and they are seeking to determine how few genes are necessary to produce a viable organism. Probably only need less than 400 genes for free-living organism.

Question 41

Simplicity vs Complexity

Answer 41

All cells now living have evolved for same length of time, including mycoplasma. Yet, why are mycoplasma relatively simple in organization, and higher eukaryotes (such as cabbages) so complex? Balance between benefits of simplicity versus value of sophistication: e.g. mycoplasma versus much more complex bacteria such as E. coli versus single celled eukaryotes (such as yeast) versus human beings. By the way, humans only possess approximately 24,000 genes, which is quite a bit more than mycoplasma, but not extraordinarily more than a yeast cell (approximately 6,300 genes).

Question 42

Prokaryotes

Answer 42

1. “Prokaryotes” meaning eubacteria and archaea (all highly evolved)- structurally simple, metabolically complex. Occupy immense variety of evolutionary niches- hot springs, deep oceans, artic tundra. By virtually any criteria: mass, diversity, or abundance, these microbes are the dominant form of life on earth. Many live quite successfully on or in us (in fact, more E. coli cells in your body than human cells, and they don’t pay tuition and had you buy them breakfast, so who is more clever)?
   a. Bacterial are generally composed of a plasma membrane (one or two bilayers thick), surrounded by a cell wall which provides protection and osmotic stabilty.
   b. No high level organization of their internal structure- certainly non-homogenous, but no true membrane-bound organelles visible.

Question 43

Eukaryotes

Answer 43

a. Defined by presence of nucleus. Essentially an organelle evolved to replicate genome and efficiently transcribe DNA into RNA. Nuclear envelope is a double layer of membrane containing specialized pores which permit mRNA to exit and proteins to enter. DNA genome packaged by proteins into structure called chromatin.
b. Share, with bacteria, a plasma membrane, but animal cells lack a cell wall (plant cells contain one made up largely of cellulose, not the peptidoglycan of the typical bacterial cell wall). Plasma membrane acts as permeability barrier and information interface, will discuss in greater detail later.
c. Unlike bacteria, eukaryotes also contain internal membrane structures- endoplasmic reticulum and Golgi apparatus. Essentially sacs, sheets, vesicles and tubes of membranes that extend through cytoplasm. These membrane structures are highly dynamic, and specialize in synthesis, modification, and transport of lipids, membrane proteins, and secreted proteins. In part, an answer to the volume versus surface area problem. Volume proportional to cube of diameter, surface area proportional to square. Thus, large eukaryotic cells have much more volume than surface area, yet must continually exchange metabolites with outside. Solution: the development of internal membranes which continually exchange with PM through exo- and endocytosis.
d. Most eukaryotes also contain mitochondria in cytoplasm.

Question 44

MItochondria

Answer 44

Most eukaryotes also contain mitochondria in cytoplasm. These are double membraned compartments devoted to oxidative phosphorylation and energy production. Probably represent remnants of an endosymbiote- a bacterium, long ago engulfed by host cell (the host was probably an archaea), which now specializes in energy production (note that eubacteria utilize their PMs for respiratory energy generation). Some controversy as to precise origins and metabolism of the original endosymbiont, but consistent with the overall hypothesis, mitochondria contain their own DNA genomes, primitive transcriptional machinery, and translational machinery. Mitochondria replicate themselves during cell division. Example of eukaryote without mitochondria- Giardia (diplomonad), but this probably represents an organism that lost its existing mitochondria, rather than a primordial form.

Question 45

Lysosomes and Peroxisomes

Answer 45

Lysosomes and peroxisomes. Also are lipid-bounded vesicles. Lysosomes contain hydrolytic enzymes involved in turnover and degradation of cell components no longer needed (obvious benefits in physically sequestering these dangerous activities). Lysosomes probably are related to large “vacuoles” in yeast. Peroxisomes contain oxidative enzymes that generate and destroy hydrogen peroxide (oxygen is quite poisonous) and are involved in lipid metabolism.

Question 46

Cytoskeleton

Answer 46

Less obvious under light microscope, but critical- cytoskeleton. An array of protein filaments and networks that give cell its shape, provide support for cell movement, allow specific organization of organelles, and assist in mitosis and meiosis. Three major kinds: microtubules, actin filaments, and intermediate filaments. These have associated molecular motors. Microtubules are also critical for cell motility as components of cilia and flagella, and actin is crucial for muscle function.

Question 47

Plants

Answer 47

Plants- in addition to these other organelles, plants have chloroplasts (outer membrane, inner membrane, with thylakoid disks) devoted to photosynthesis. In common with mitochondria, chloroplasts probably represent an ancestral symbiosis created, in this case, by engulfment of a photosynthetic bacterium. Chloroplasts contain own genomes, transcriptional and translational machinery which resemble closely those of eubacteria.

Question 48

In light of complexity, what is critical for both eukaryotes and prokaryotes

Answer 48

Regulation

Question 49

Regulation in single celled organisms

Answer 49

For single celled organisms, important elements of regulation are metabolic in nature- as external environment changes, single cell organisms must respond to it in appropriate way. Frequently reflected at level of gene expression. Example- changes in carbon source availability in bacteria, or availability of phosphate in yeast. Single cells have evolved into highly complex, very sophisticated entities, such as protozoa.

Question 50

Regulation in multi-cellular organisms

Answer 50

Yet, one additional, viable evolutionary turn appears to have been the generation of multicellular organisms. Here, regulation becomes increasingly focused on coordinating the actions of the multiple cells which make up the organism, rather than a response to the outside environment.

1. All organisms are multicellular at some point in replication, some (yeast, bacteria) can form quite elaborate chains of cells. Thus, simplest level of multicellular organisms is formation of a colony of identical cells (e.g. Gonium- flat disks of identical photosynthetic cells).
2. Critical step- specialization. Not all cells in colony identical.

Question 51

Light Microscope

Answer 51

Light microscopes- quite remarkable. Simple solid-state lens requires no power (except for illumination), yet can magnify up to 1000x. Resolution limit of unaided eye= 100μm (1/10 millimeter). Typical animal cells are 10 to 20 μm in diameter, typical bacteria are 0.5 to 1 μm.

Question 52

Resolution of Light Microscopes

Answer 52

Resolution of light microscopes is limited by wavelength of light, to about 0.2 μm (note a hydrogen atom is approximately a thousand times smaller). Bacteria and mitochondria are among the smallest objects whose shapes can be directly visualized by light microscopes.

Question 53

Constraints of Light Microscopes

Answer 53

Other constraints- most biological material are thick and colorless. Must be fixed, thin sectioned to single cell thicknesses and/or stained to use in microscope.

Question 54

Visible stains in light microscope

Answer 54

Visible stains extremely powerful approach for differentiating different types of cells and different subcellular components (e.g. hematoxylin stains RNA, DNA, and negatively charged proteins). Critical for clinical pathologists (e.g. cancer cell detection

Question 55

Fluorescent stains in light microscope

Answer 55

Fluorescent stains- variation of same concept, but instead of absorbing light, emit it. Excite fluorophore at one wavelength, it will fluoresce at a longer wavelength. Very sensitive, by using correct filters on microscope can detect very small quantities of stain/dye. Can use in a similar fashion as visible light dyes (e.g. propidium iodide very specific for DNA),

Question 56

What do most dyes require

Answer 56

Many (but not all dyes) require cell to be fixed (i.e. killed). Thus, cannot always follow
a process in real time.

Question 57

Phase contrast or Nomarski-optics

Answer 57

Phase contrast or Nomarski-optics microscopes rely on changes in refractive index of different cell components to create an image in absence of dye. Useful for following living processes. Can be further enhanced using electronic image processing

Question 58

Thick samples and light microscopes

Answer 58

Above methods (dyes and such) still unable to visual thick samples, which produce multiple images stacked and distorted above one another. Classically, one solution is microtome- a method of slicing samples very thinly. Yet, even at this level one losses resolution. Recent approach- confocal scanning and deconvolution microscopes, which can avoid this problem by restricting excitation of a fluorescent image to a single plane within the cell, using special apertures and lenses, or by use of electronic data processing of a series of images at different focal planes, respectively.

Question 59

Electron microscopes

Answer 59

Electron microscopes. How to avoid inherent limit of light microscope resolution (0.2μm)? Might use a form of radiation with shorter wavelength. X-rays and gamma rays: actually some progress in this area, but technically difficult to use and to focus. Physics demonstrates that there is a dual wave/particle nature to matter. Accelerated electrons can be diffracted in a manner analogous to photons, and can be focused using magnets instead of lenses. Greater the energy, shorter the wavelength. Current EMs have practical resolution limit at best of 0.1 nm, and in practice, approximately 2 nm (100 time better than optical microscopes). Two main types- transmission EM versus scanning EM.

Question 60

Limitations of Electron microscopy

Answer 60

Same limitations in sample preparation as light microscopy- most samples must be fixed and stained (most biological materials are transparent to electrons, stains often use heavy metals like uranium and lead) and exposed to a vacuum (to avoid electron scattering by air). Thus, most EM is performed on dead material, cannot directly follow living processes. Transmission EM has highest resolution, but requires samples to be thin sections. Scanning EM has lower resolution, but can be performed on three dimensional objects. sometimes living cells, and provides very good surface definition.

Question 61

Atomic Force Microscopy

Answer 61

Atomic force microscopy. Simple concept- a very thin probe is dragged across the surface of the specimen and irregularities in the surface are measured electronically and computer decoded into a image. Thus, can actually detect single large atoms by this method. Currently, using biological materials, can resolve proteins bound to DNA as a lump on a string; improvements in this method are likely to continue. Note however that AFM is only usable on fairly pure materials in a test tube, and is not typically suitable for whole cells or complex cell extracts.

Question 62

X-Ray, EM Diffraction, NMR

Answer 62

X-ray and EM diffraction small angle X-ray scattering, and NMR. Only techniques that provide detailed information as to three dimensional structure of biological molecules. X-ray and EM diffraction uses math (Fourier transforms), rather than lenses, to get around inability to focus X-rays; both require crystalline or para-crystalline arrays, thus mostly restricted to pure molecules, and relatively small proteins or DNAs. NMR measures distances between different atoms in a macromolecule, lower resolution than X- ray diffraction, but can study purified proteins in concentrated solutions. SAXs does not require crystals.

Question 63

Cell Culture

Answer 63

Cell culture

1. Some, but not all cell types can be grown in culture in appropriate media. Provides method of studying cells over time, and allows manipulation of conditions, addition of specific metabolic precursors, study of single cell phenotypes.
   a. Bacteria were the first organisms for which defined culture conditions were achieved. Bacterial culture is generally simpler than that of eukaryotic cells; bacteria can make many required substances themselves from simple precursors, and are not subject to complex regulation of cell growth displayed by multi-cellular organisms. Nonetheless- many bacteria have yet to be cultured in laboratories.
   b. Animal and plant cells- more complex culture conditions: require externally supplied, complex organic molecules for growth- sugars, amino acids, salts, vitamins (e.g. functions normally provided by liver). But frequently, even these only keep cells stay alive, not sufficient for replication. Must provide missing clues that in whole organism regulate growth and proliferation. e.g. growth factors (insulin, transferrin, etc). Some cells can be manipulated genetically to grow in culture which otherwise would not (add telomerase or growth promoting gene). Recent progress in creating stem cells that grow in culture and can be induced to form all or most of the cell types seen in a whole organism. However, many kinds of cells still cannot be cultured successfully. Also, even those that do grow in culture may display a partly or highly abnormal phenotype.

Question 64

Cell and Molecule fractionation

Answer 64

1. Can fractionate cells into different components to study individually in test tube:
   a. Into different organelles- lyse cells and separate different organelles based on their size and density. Very valuable for dissecting functions of these organelles.
   b. Into supramolecular complexes that perform specific functions- e.g. translation, transcription. These in vitro systems permit the conditions and components to be varied and have played key roles in experimental dissection of these systems.
   c. Into single molecules. Chromatography (ion exchange, gel-filtration, affinity). Electrophoresis (size versus charge). Permit actions and structures of individual molecules to be studied in detail. Examples:
   i. Enzymatic activity of proteins and certain RNAs can be studied in pure systems
   ii. Sequence of proteins and nucleic acids can be determined. e.g. matrix-assisted laser desorption-time-of-flight (MALDI-TOF) spectrometry
   iii. Three dimensional structures can be defined by X-ray crystallography and nuclear magnetic resonance spectroscopy.

Question 65

How are cells, and cell function, studied

Answer 65

1. Microscopes
2. Cell culture
3. Cell and molecule fractionation
4. For nucleic acids- genetics provides means of fractionation distinct from the physical/chemical approaches described above- molecular cloning through use of recombinant DNA.
5. Tracing molecules in cells

Question 66

Tracing molecules in cells.

Answer 66

1. Use of radioactive precursors- radioactive molecules can be identified with extremely high efficiency at very low concentrations. Radioactive labeling can restrict radioactivity to particular type of molecule (e.g. radioactive amino acids principally label proteins) and can distinguish between chemically identical molecules with different histories (e.g. freshly synthesized proteins versus old proteins). Can follow radioactivity by autoradiography, or by following the position of the radioactivity in any of the fractionation methods described above.
2. Use of specific antibodies to proteins of interest. Both for immunofluorescence/EM, or to trace molecules during physical fractionations described above.
3. Ion concentrations (H+, K+, Ca++, etc) can be measured in living cells with selective microelectrodes. Extremely fine glass needles can be poked into cell (membrane seals around) to study concentrations of specific ions, flows of ions across the membrane, or can even be used to introduce specific molecules into the interior of the cell.
4. Certain ion-sensitive fluorescent or luminescent molecules can be used to study ion fluxes in living cells. e.g. aequorin is a protein that emits light in presence of free Ca++. Can thereby detect an influx of Ca++ upon egg fertilization.
5. Nucleic acids can be followed by nucleic acid hybridization. In situ hybridization allows the location of specific nucleic acids to be visualized by fluorescent light microscopy. Can even “see” specific gene’s location within interphase nucleus, or path mRNA takes on way out of cell (caution: this provides less detailed information than you might think). In vitro nucleic acid hybridization permits nucleic acids fractionated by electrophoresis (or more rarely by chromatography) to be identified.
6. Recently, methods such as DNA microarrays and RNA sequencing study the levels of multiple mRNAs simultaneously in a cell population. Can be used to understand global changes in gene expression in response to different event

Question 67

Privalsky Uncertainty Principle

Answer 67

Privalsky uncertainty principal: The more accurately a biological process can be studied, the less physiologically relevant the conclusion, and visa versa.

Question 68

Plasma Membrane

Answer 68

Plasma membrane- defines the cell- separates self from environment, is site of exchange of solutes (metabolites, catabolites, gases) with outside. Is also an information interface for interactions with other cells and informational molecules in environment (plasma membrane contains receptors for this external information). In bacteria PM is also site of oxidative phosphorylation

Question 69

Lipid Bilayer

Answer 69

Universal basis for cell membrane structure-all phyl

Question 70

Lipids are what kind of molecules

Answer 70

Lipids are amphipathic molecules, insoluble in water but highly soluble in organic solvents. Have polar end (e.g. phosphate, sugars) and hydrophobic end (usually fatty acids between 14 and 24 carbons long, can be saturated or unsaturated). In water, lipids tend to spontaneously form
micelles and self-sealing bilayers. PM of average cell has about 109 lipid molecules. Lipids in bilayer include phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and cholesterol. Precise lipid composition can vary from cell type to cell type, or under different conditions (e.g. different temperatures), resulting in changes in the physical properties of the lipid bilayers (fluidity, charge, etc).

Question 71

Two-dimensional fluidity of lipid bilayer

Answer 71

Lipid bilayers behave as two-dimensional fluids: in absence of constraints, lipids on each face of bilayer can diffuse rapidly in plane of membrane (in 1 sec can complete circumnavigation of entire outer surface of a bacterial cell), but unaided, lipids rarely can flip-flop from one surface of bilayer to the other (would require burying hydrophilic group at some point). Special proteins in living cells (phospholipid translocators) assist in transferring lipids from one surface to the other during synthesis of membranes.

Question 72

Symmetry of the lipid bilayer

Answer 72

The lipid bilayer in living cells is asymmetrical- in part generated by the nature and different efficiencies of the phospholipid translocators present in the ER. For example, in red blood cell, high levels of choline lipids are in outer layer, high levels of primary amino group lipids are in inner layer. Glycolipids (5% of total lipids) are found on the outer surface of all animal cell plasma membranes; some have quite complicated oligosaccharide structures. Role may be protective, or to affect the electrical properties of the lipid bilayer, or may be involved as recognition molecules in cell to cell interactions.

Question 73

How do proteins associate with membranes

Answer 73

Proteins can associate with membranes in different fashions, but in all cases appear to do so by use of hydrophobic domains. Average soluble protein will not dissolve in or cross a lipid bilayer.

Question 74

Integral membrane proteins

Answer 74

a. integral membrane proteins are embedded within membrane itself, generally can only be removed by disrupting the lipid bilayer itself with detergents or organic solvents. Integral membrane proteins can transverse (transmembrane proteins) or penetrate deeply the entire bilayer; these have exposed hydrophobic amino acids which interact with lipid side chains, and hydrophilic amino acids which typically interact with polar lipid and water molecules on each face of membrane.
b. integral membrane proteins can also be associated with either face of the lipid bilayer, perhaps anchored to membrane at one end, but with rest of protein is fully exposed to aqueous layer.
i. sometimes, this anchor is a fatty acid chain that is inserted in the cytoplasmic side of the lipid bilayer, and is covalently attached to an amino acid in the protein. e.g. myristic acid or prenyl groups help bring some important molecules involved in cancer causation into proximity of plasma membrane
ii. similarly, outer surface proteins can be anchored by linkage to glycosylphosphatidylinositol molecules
iii. some proteins contain both transmembrane hydrophobic amino acid domains and are further stabilized in the membrane by covalent attachment of lipid.

Question 75

Peripheral membrane proteins

Answer 75

peripheral membrane proteins are associated with membranes more weakly, frequently through non-covalent interactions with integral membrane proteins. These are more easily removed by treatment with high salt

Question 76

Common methods for proteins to cross the lipid bilayer

Answer 76

2. It is common for transmembrane proteins to cross the lipid bilayer as an α-helix region. This helps maximize hydrogen bonding in absence of water. Transmembrane proteins can cross membrane once with one α-helix segment, or multiple times with multiple helical segments. Need an α-helix 20 to 30 amino acids long to fully cross a lipid bilayer once.
3. Another method for protein to cross membrane is as ß-barrel, a ß-sheet structure which helps orient the hydrophobic side chains facing out toward the hydrophobic lipids in the membrane.

Question 77

Protein’s similarity to lipids

Answer 77

. Implicit in all this: proteins, like lipids, are inherently asymmetrically distributed in membranes. Also- many membrane proteins surround themselves with specific kinds of lipids.

6. Like lipids, many proteins can diffuse laterally in plane of membrane. Also can rotate in place, but do not usually spontaneously flip-flop. Cells however can confine particular proteins and lipids to defined domains of a membrane. For example, in kidney the cells are polarized so that a given solute carrier is on a specific side of the cell, and solutes therefore travel in one direction. The proteins present on one surface of these cells do not exchange with the proteins found on the other surface; specific barriers exist to achieve this (e.g. tight junctions; specialized cell-cell contacts that also bar membrane protein diffusion). Another method is to specifically anchor the membrane proteins to particular internal or external components of the cell (e.g. cytoskeleton or extracellular matrix).
7. Like lipids, many proteins exposed to cell surface are glycosylated (have covalently joined sugars). Very diverse glycoprotein and glycolipid structures are possible. Glycosylation may play important protective roles or serve in cell-cell recognition. For example, white blood cells are able to target to regions of inflammation to combat infection in part due to the induced synthesis of P-selectins at the inner surface of the blood vessels at the site of inflammation. This selectin is a protein which binds to a specific oligosaccharide present on the glycolipids and glycoproteins of white blood cells, stopping their circulation in blood and inducing the white blood cells to pause and then migrate out into the site of infection.

Question 78

Diffusion

Answer 78

diffusion- some hydrophobic or very small hydrophilic molecules can cross membranes by simple diffusion. Examples- oxygen, carbon dioxide, and to a lesser extent, water.

Question 79

Facilitated transport

Answer 79

b. facilitated transport- larger polar or even small but highly charged molecules pass across lipid bilayers inefficiently on own. e.g. sugars, amino acids, proteins, nucleic acids, ions such as sodium, potassium, calcium, chloride, etc. Facilitated by specific proteins which aid in transporting these molecules across lipid layer. already discussed one example- phospholipid transporters.
c. facilitated transporters come in two major flavors-
i. carrier proteins which bind the specific solute to be transported and undergo a series of conformational changes to transfer the bound solute across the membrane, versus
ii. channel proteins (or “pores”) which need not bind the solute, but form hydrophilic pores across membranes (much faster, but frequently much less specific than carriers).
d. facilitated transport by carriers can be passive (i.e. down a concentration gradient until both sides equilibrate) or active (by coupling to energy source can actually pump solutes up a concentration gradient). Energy can come from ATP hydrolysis, other solute gradients, or light (e.g. bacterial rhodopsin).

Question 80

Carrier proteins

Answer 80

i. carrier proteins which bind the specific solute to be transported and undergo a series of conformational changes to transfer the bound solute across the membrane, versus

Question 81

Channel proteins

Answer 81

ii. channel proteins (or “pores”) which need not bind the solute, but form hydrophilic pores across membranes (much faster, but frequently much less specific than carriers).

Question 82

ABC Transporters

Answer 82

ABC transporters- are the largest known protein family. Involved in ATP- coupled transport of many different compounds in different biological phenomena. Include multidrug resistance protein, that pumps chemotherapeutic drugs out of cancer cells; similar pumps can confer resistance to drug treatment on parasitic protozoa.

Question 83

Ion channels

Answer 83

i. -ion channels can, when open, transport up to 100 million ions per second, but rely on passive transport (cannot be coupled to energy). The size, shape and charge on the pore side of the channel determines the ion selectivity.
ii. -most ion channels are regulated in some fashion (gated). Three major kinds- voltage gated, mechanically gated, or ligand gated.
iii. Typically consist of transmembrane protein assemblies that create a pore in the membrane, plus some sort of selectivity “filter” which determines the nature (charge and size) of the ion that can be transported. Note that the filter can even allow larger ions to be transported (e.g. potassium) and filter out smaller ones (e.g. sodium) by providing the correctly spaced carbonyl side chains to compensate for loss of hydration as ion enters pore.

Question 84

Electrical properties

Answer 84

i. -ions have charge. Thus, every ion that crosses a membrane without a counterion establishes both a chemical gradient and an electrical gradient (or voltage) across a membrane. Lipid bilayers are electrical insulators.
ii. -Na+/K+ pump actually develops a strong electrical gradient (membrane potential) in two ways. First of all, pump actually pumps out three Na+ for every two coming in (i.e. electrogenic). In addition. there are K+ leak channels that permit some of K+ to flow out of cell, without counter-ion, and without equivalent flow of Na+ in. Thus, most animal cells are negatively charged inside versus outside. Most animal cells have a membrane potential on average of 1/10 volt.
iii. -nerve cells make special use of this membrane potential, and voltage gated channels, to conduct nerve signals. Will discuss in detail later, but basic idea is that a transient influx of Na+ at one side of elongated nerve will drop the voltage, causing adjacent voltage-gated channels to let in more Na+, which repeats the process until a wave of depolarization sweeps from one end of the nerve to the other, in essence an electrical signal comparable to a telephone wire (though ions, and not electrons, move).

Question 85

Na+ gradient

Answer 85

Most cells use the Na+ gradient in this same fashion to regulate intracellular pH. e.g. Na+/H+ exchanger uses antiport of Na+ in to pump H+ out. In fact, both photosynthesis and oxidative phosphorylation synthesize ATP from ADP through a H+ gradient intermediate that runs a pump-like exchanger backward.

Question 86

The Na+/K+ gradient (concentration gradient…)

Answer 86

concentration gradients can actually store energy. The Na+/K+ gradient is used to help indirectly drive the transport of other molecules by sym- or anti- porters. e.g. in symport, Na+ binds to outside of carrier together with a sugar or amino acid, Na+ flowing into cell down its concentration gradient drags along the sugar, opposite the sugar concentration gradient.
iii.-Most cells use the Na+ gradient in this same fashion to regulate intracellular

Question 87

three distinct ways things get from one subcellular compartment (e.g. site of synthesis) to another (e.g. site of action):

Answer 87

1. transmembrane transport. Will discuss first. This is process by which membrane and secreted proteins (synthesized on cytoplasmic surface of ER) pass through ER membrane to associate with ER lumen. Also-phospholipid translocators fit in this category.
2. vesicular transport. The lumen of the ER is not the final destination for most proteins or lipids. Some proteins end up permanently residing in plasma membrane (PM), some are released into extracellular environment, some target to lysosomes. Transferred by exchange of small hollow balls of lipid and protein
3. gated transport- method by which RNA leaves nucleus and proteins enter. Distinct from (1) in that selective pores/transporters in nuclear membrane control this process, allowing free diffusion of some molecules and regulated gating of others.

Question 88

Endoplasmic Reticulum

Answer 88

1. Typically a net-like complex of branching tubules, sacs, and vesicles extending through much of the cytosol, probably forming a continuous or near-continuous sheet enclosing an internal space called the ER lumen. Over half of the membrane in a typical animal cell is in ER.
2. The lumen typically may represent 10% of total internal volume of cell, and as previously mentioned, is topologically equivalent to extracellular space.
3. ER plays the central role in lipid biosynthesis. It is also the site of synthesis of most integral membrane and secreted proteins, as well as most of the proteins which make up the ER itself, the Golgi apparatus, and the lysosomes.
4. Morphologically, the ER consists of rough ER and smooth ER. Rough ER appears so because of the presence of large numbers of ribosomes associated with cytoplasmic surface of membrane in these region. Smooth ER is responsible for transit of proteins to Golgi, for lipid synthesis, for aspects of lipoprotein synthesis, and is location of a number of detoxification enzymes (e.g. cytochrome P450s). Can also sequester Ca++ in certain cell types.

Question 89

Rough ER

Answer 89

The rough ER is the site of membrane and secreted protein synthesis.

1. In essence, the rough ER captures proteins destined to be integral membrane or secreted proteins as they are synthesized.
2. All protein synthesis initiates on functionally identical ribosomes in the same manner. The initiation codon is identified on the mRNA during the formation of a pre-initiation complex with (in eukaryotes) the 40s ribosomal subunit. The 60s ribosomal subunit then associates, and the protein begins to be synthesized from N-terminus to C-terminus.
3. If protein is destined to be cytoplasmic, the process simply continues, the ribosomes translocating the mRNA remain soluble in cytoplasm, and mature polypeptide chains are released when ribosome encounters the translational terminator.
4. In contrast, proteins destined to enter ER contain an ER signal sequence on or near their N- terminus. This sequence is typically a string of hydrophobic amino acids 8- or more long. Individual sequence can vary, but all known ER signal sequences are hydrophobic in nature. This nascent polypeptide chain is bound by a specialized factor called the signal-recognition particle (SRP). Association of the SRP with the nascent chain is usually co-translational, and in fact, arrests further translation of the protein.
5. This pause in translation gives the mRNA/ribosome/nascent polypeptide/SRP complex time to associate with the rough ER, thereby preventing accidental continued translation and release of the protein inappropriately into the cytoplasm. Intriguingly, the SRP is a large complex containing both proteins and an internal RNA call the SRP RNA. The SRP- RNA may facilitate interactions of the SRP with the ribosomal RNAs, and/or may be a catalytic relic of the RNA world.
6. This complex now associates with a protein exposed on the cytoplasmic side of the rough ER called the SRP-receptor. Association with the SRP-receptor results in several events. The ribosome/mRNA/nascent protein complex is handed off to a ribosome receptor/protein translocator complex (Sec 61 complex), and the SRP dissociates. This permits the arrest of protein translation to be released, and the nascent protein is led down through the protein translocator through the ER membrane into the lumen of the ER. The ribosome is now immobilized on the cytosolic side of the ER (ribosome actually seals the top of the channel), and the protein is threaded through the membrane pore as it is translated. Thus, the ER signal peptide also serves as a start transfer signal (and is actually held by the translocational machinery as the rest of the protein enters the ER lumen). Pore closes once the protein is fully translocated into the ER lumen.
7. Energy for this process not yet fully understood. Much of force to thread protein through membrane co-translationally appears to be generated by the translational machinery/ribosome itself. In bacteria, a SecA translocation motor helps push the protein through the membrane. In eukaryotes, a BiP chaperone on the lumen side helps pull the protein through the membrane. Both use ATP as energy. On the other hand, there are a certain proteins which can translocate into the ER lumen after they are fully synthesized and released from the ribosome. Thus, at least in these cases transit into the ER lumen is driven by a separate active transport process, utilizing ATP as a source of energy for accessory proteins that bind the polypeptide and either push (bacteria) or pull (eukaryotes) it through the Sec61 channel.
8. Note then that ribosomes associate with the rough ER only after they begin to synthesize ER- targeted proteins, and that the ER-association is transient, and the very next protein synthesized by that particular ribosome may very well be a cytoplasmic protein.
9. The passage of the polypeptide chain through the membrane occurs due to the presence of an aqueous pore associated with the protein translocator complex. The pore opens when the nascent polypeptide associates with the protein translocator (is apparently gated by the signal sequence) and closes after the protein is complete.
10. The protein is typically threaded through the channel in an unfolded state; folds properly on lumen side of membrane.

Question 90

What determines a protein’s release

Answer 90

The fate of the signal peptide, and the location of other membrane anchoring domains in the protein, determine its release as a soluble protein, or its topology as a transmembrane protein.

Question 91

Post-translational modification

Answer 91

D. Post-translational modifications. Many proteins are post-translationally modified once they enter the ER. Already gave example of proteolysis of N-terminal signal peptide. The rough ER is also the site of many of the core glycosylations that take place on proteins that are destined to become glycoproteins.

1. N-linked glycosylations take place on specific asparagine residues in proteins.
2. a “core” oligosaccharide is first synthesized in a step by step fashion on a special dolichol phosphate lipid scaffold oriented toward the cytoplasmic face of the rough ER. This oligosaccharide core is typically composed of many mannose sugars, with additional glucose and N-acetyl glucosamine residues. The enzymes which create this structure are called glycosyl transferases, and use nucleoside diphosphate-sugars precursors.
3. The core oligosaccharide-dolichol lipid is next flipped to the lumenal face of the rough ER and transferred en mass to the asparagine of the target protein. These modifications take place in the rough ER, can occur cotranslationally.
4. This core oligosaccharide is next further modified in the ER. Additional glucose and mannose residues can be added by transfer from monosaccharide dolichol lipids (perhaps a total of 14 saccharides long). Other core sugars initially transferred en bloc from the dolichol phosphate intermediate are trimmed off the glycosylated protein.
5. In most cases, the glycoprotein will be transferred from rough to smooth ER, and ultimately to the Golgi for further processing of the oligosaccharides prior to the final destination (PM, secretion, or a subcellular organelle). Will discuss these processes in next lecture.
6. Part of role of protein glycosylation is to mark incompletely folded proteins for the actions of chaperones called calnexin and calreticulin, which recognize proteins with single terminal glucoses (which are cycled on and off until protein is fully folded). Improperly folded proteins are exported from ER and degraded in cytosol.

Question 92

Synthesis and assembly of membrane lipids themselves

Answer 92

A. Most assembled enzymatically on the cytoplasmic surface of the ER, where the necessary substrates are found.
B. Notably, because de novo lipid synthesis therefore takes place on cytoplasmic surface of ER membrane, phospholipid transferases have to catalyze the flip of these lipids to the lumenal half. “Scramblases” are relatively non-specific (random) in flipping lipids, whereas the more narow specificity of “flippases” contributes to the asymmetry in the lipid compositions of the inner and outer faces of the membrane.
C. ER also produces cholesterol and the precursors for sphingolipids. Golgi is primary location where sugars will be added to certain lipids to make glycolipids.
D. Plasma membrane, Golgi apparatus, lysosomes, and endosomes all form part of a membrane system that communicates with the ER by transport vesicles (will be discussed next). These transport vesicles exchange both newly synthesized proteins, and lipids with these other compartments. Mitochondria, plastids, and peroxisomes obtain their lipids from ER through actions of water- soluble carrier proteins called phospholipid exchange proteins.

Question 93

The Golgi apparatus

Answer 93

1. Major hub and switching station
2. Major site of further glycosylation of proteins and lipids initially glycosylated in the ER
3. Golgi also major site of synthesis of polysaccharides destined for extracellular deposition in plant cell walls and of the glycoaminoglycans destined for the animal cell extracellular matrix.
4. Consists of a series of stacks of flattened membrane cisternae and many tiny vesicles.
5. Functionally and structurally divided into a cis Golgi (initial location for entry of proteins and
   lipids passing through Golgi), a medial Golgi, and a trans Golgi (exit surface).
6. Golgi apparatus particularly large and prominent in cells specializing in secretion- e.g.
   pancreatic cells.

Question 94

Transport from ER to Golgi

Answer 94

1. Vesicles containing ER membrane, membrane proteins, and ER lumen contents constantly bud off a specialized region of the smooth ER (lacks ribosomes). This vesicle budding appears to have both selective and non-selective aspects. Many proteins have specific ER exit signals and are efficiently budded off ER and transported to Golgi. Exact signal sequences not clear- some exit signals may be in the form of a specific oligosaccharide. Other proteins in ER without specific signals, can still be budded and transported to Golgi in a more general but less efficient “bulk-flow.” Incorrectly folded protein remain in the ER until they fold properly, or they are degraded. A quality control checkpoint.
2. Vesicles budded from ER can fuse with one another to form a vesicular tubular cluster that is moved by motor proteins along microtubular tracks toward the cis-Golgi where the cluster fuses and delivers its contents into Golgi.
3. Yet, what of the proteins that are meant to stay in the ER (e.g. some chaperones, some glycosyl transferases, etc?). These have specialized sequences (e.g. many ER soluble proteins have KDEL sequences, many ER membrane proteins have KKXX sequences) that form an ER retention signal. These proteins often leak out of the ER and enter the Golgi along with everyone else, but are then retrieved back from the cis-Golgi into the ER by specialized recovery vesicles in an endless loop. Thus the KDEL and KKXX sequences are really a retrieval, not a true retention sequence. These act together with additional tethering interactions that slow or prevent the exit of other ER resident proteins from ER.
4. Some drugs are known which affect these processes. Brefeldin A appears to block the forward transport from ER to Golgi without affecting the reverse transport. After Brefeldin A treatment, the Golgi actually appears to be mostly retrieved into the ER, collapsing the Golgi apparatus and mixing its contents with those of ER.
5. Similarly, there is also a bulk flow of lipids from ER to Golgi.
6. Transport from cis to median to trans Golgi is controversial- some believe this is largely by shuttle vesicles like ER to cis Golgi, others believe that the large cisternae of Golgi themselves mature and move through the Golgi apparatus, taking contents with them.

Question 95

Protein processing in the Golgi

Answer 95

1. N-linked oligosaccharides have already been added to glycoproteins in the ER as a single block, and some of these sugars have already been trimmed off. Although some of these proteins retain the high-mannose core oligosaccharides first synthesized in the ER, further, often complex modification of these oligosaccharides can occur in the Golgi. More trimming, step-by-step addition of additional carbohydrates by specific glycosyl transferases. Extent of the glycosylation can be used as a measure of the location of a protein (ER vs Golgi in the secretory pathway (endo H cleaves only unprocessed, high- mannose forms that typically have not entered the Golgi yet).
2. Soluble glycoproteins destined for lysosomes are phosphorylated on mannose sugars in the Golgi. Some oligos sulfated. Also some proteins have short oligosaccharides added to threonine and serine amino acids (O-linked glycosylation); this too occurs in the Golgi.
3. The different steps in processing appear to be spatially organized in the Golgi, with enzymes responsible for the early processing found in the cis Golgi, and enzymes responsible for the late processing found in the trans Golgi.
4. Note that the sugars are all added to the lumen side of the proteins (topologically equivalent to the outside surface). N-linked glycosylation is unknown for cytoplasmic proteins.
5. What does N-linked glycosylation do? Unique to eukaryotes, but function remains incompletely understood. Some ideas:
   - May make certain proteins and protein domains more hydrophilic or otherwise change the surface properties of these proteins. Help in folding.
   - May inhibit degradation by proteases, or in parasites, alter the immunological properties
   - May have regulatory or recognition properties- large number of possible sugars, and myriad ways of linking together, generate an immense diversity of oligosaccharide chains that could play important recognition roles. e.g. selectins.

Question 96

What is the default pathway for lipids and many proteins

Answer 96

trans-Golgi to PM/cell surface is the default pathway (in absence of any other signal) for lipids and many proteins. Once vesicles fuse with PM, the vesicle membrane proteins and lipids mix with those already in PM, the soluble proteins are released into the extracellular environment.

Question 97

In regulated secretion the secreted proteins are where?

Answer 97

In regulated secretion the secreted proteins are stored in secretory vesicles until the proper moment for release.

a. secretory products are packaged into appropriate vesicles in trans Golgi. Exact signal unclear, probably a patch of amino acids.
b. after secretory vesicle buds off trans-Golgi, contents become acidified by actions of an ATP-dependent H+ pump, and extremely concentrated, in part, by retrograde transport of contents not destined for exocytosis.
c. often the secretory proteins are proteolytically processed at this point as well. (pro-proteins need to be proteolytically cleaved to be active). This keeps hydrolytic enzymes inactive until release from cell. Also the presence of a large protein precursor can in many cases allow transport and processing through ER and Golgi of polypeptides that in their final, functional forms would be too small for these processes (e.g. enkephalins, which are 5 amino acids long in mature form).
d. At least in causes of nerve cells, secretory vesicles can be transported by molecular motors quite long distances from Golgi to final site of approximation to PM. There, the secretory vesicles wait near PM until signaled to release their contents.
e. signal for release can be electrical (nerves) or hormonal, and results in fusion of secretory vesicle with PM and release of contents.
f. secreted membranes in synaptic vesicles can be rapidly retrieved, refilled, and used to reform new vesicles.

Question 98

Lysosomes

Answer 98

Lysosomes- the principal site of intracellular hydrolytic reactions and digestion

1. Bags of hydrolytic enzymes used for degradation of macromolecules, contain proteases, nucleases, glycosidases, lipases, phosphatases, and sulfatases. Essentially a recycling plant. An acid environment. Obviously advantageous to place all these nasty things in a separate membrane-enclosed environment, and to make pH optimum different from that of cytoplasm (cytoplasm is more alkaline).
2. Lysosome enzymes (typically hydrolyases) must be targeted to these compartments, as well as the unique membrane proteins which define these organelles. How? It is a side branch of the ER to Golgi to PM apparatus. In addition to these lysosomal enzymes, macromolecules which are to be degraded by these enzymes must be also targeted to the lysosomal compartment (done by multiple pathways).
3. In plants and yeast, larger structures called vacuoles (very large, central) perform the lysosomal functions. These can also serve as storage sites for nutrients and waste products, and can help control osmotic pressure.

Question 99

Many soluble enzymes destined for lysosomes are synthesized where? Diverted where?

Answer 99

Many soluble enzymes destined for lysosomes (or vacuoles) are synthesized on membrane- associated ribosomes in ER, enter ER lumen, and are then diverted from the default pathway in the trans-Golgi
1. lysosomal hydrolases are specifically phosphorylated with a mannose-6-phosphate group. This occurs in a multi-step reaction:
a. proteins destined for lysosome are N-glycosylated in ER as usual and enter the cis-Golgi with the default pathway proteins
b. a separate signal patch in the hydrolase is recognized by a cis-Golgi phosphotransferase. The phosphotransferase adds an N- acetylglucosamine phosphate to one or more of the mannose residues on each oligosaccharide chain.
c. A second enzyme, also in the cis-Golgi, then splits off the N-acetyl glucosamine, leaving the mannose-6-phosphate
d. The presence of mannose-6-phosphates on the oligosaccharides are recognized in the trans Golgi by a mannose-6-phosphate receptor that packages
these hydrolytic proteins into transport vesicles. These vesicles then target to and fuse with an immature form of lysosome called a late endosome. The mannose-6-phosphate receptor binds to the mannose-6- phosphate glycoproteins at pH 7 (i.e. trans-Golgi pH), but releases at pH In this disease, almost all lysosomal hydrolases mis-sort to secretory pathway (end up in blood), and lysosomes accumulate un-degraded substrates
2. Certain lysosomal membrane proteins and some soluble hydrolyases are sorted through a mechanism independent of mannose-6-phosphate, but this pathway also operates, at least in part, through the ER-Golgi network. Thus, these membrane- associated proteins also have signal sequences that target them, in part through vesicular transport, to lysosomes.

Question 100

How are the substrates delivered to lysosomes?

Answer 100

1. Multiple pathways
   a. phagocytosis (in specialized cells, e.g. macrophages and ameba) can engulf extracellular objects to form a phagosome, which is then converted to a lysosome as in autophagy. Often a triggered process- e.g. by antibody coating a foreign object. Involved in cleaning of wounds of bacteria, dead cells, etc.
   b. endocytosis. Basically a process by which vesicles originating with PM then shuttle proteins and other materials into cell (these endocytic vesicales are much smaller than phagosomes). These inwardly directed vesicles then meet up with the outwardly directed hydrolytic enzyme vesicles, fuse, form a late endosome, and ultimately mature into a lysosome.
   c. autophagy- used organelles can become engulfed by membranes derived from the ER, creating an autophagosome, which then fuses with a lysosome for degradation. A recycling program. In fact, also a means of targeting of certain lysosomal/vacule hydrolytic enzymes

Question 101

What is a protein coat

Answer 101

Most transport vesicles bud off of specialized coated regions (pits) of membranes, and become coated vesicles (protein coats). The protein coat provides means of driving budding and fusion, and a means of distinguishing one kind of vesicle from another.

Question 102

Three major kinds of coats

Answer 102

i. Clatharin-coated vesicles (involved in transport of transmembrane proteins from trans-Golgi and from plasma membrane, for example). Clatharin consists of three large and three small protein subunits which associate to form a three-legged structure called a triskelion (very Star-Treky). These triskelions associate with a defined region of the donor membrane and spontaneously form into polyhedral cages or baskets, and thus (together with interactions with adaptins, see below) drive formation of a budding membrane vesicle. Dynamin associates with the narrow neck of the developing vesicle and, using GTP hydrolyses, pinches it off. Actin may also contribute. Once the vesicle pinches off, the clatharin cage is quickly dissociated and the vesicle uncoats. Uncoating is driven by ATP hydrolysis. Initial recognition of membrane patch by clatharin is probably through adaptins, proteins which associate with the incipient bud by contacting cytoplasmic domain of cargo receptors. Thus, specific cargo molecules are trapped by cargo receptors inside the lumen of the donor compartment. The transmembrane cargo receptors are recognized by specific adaptins, thus providing an identifying mark on the cytoplasmic face for clatharin association. Different adaptins are associated with targeting to different acceptor membranes.
ii. COPI and
iii. COPII (coatmer)-coated vesicles are associated with vesicular transport from early in the secretory pathways: COPII coated vesicles bud from ER; COPI vesicles bud from pre-Golgi and Golgi cisternae. Coatmers are formed of seven different proteins (including some that resemble clatharin adaptins). Coatmer- coated vesicles do not form spontaneously, but require ATP for formation, and do not disassemble after synthesis, but remain

Question 103

How is specificity achieved in finding and fusing with acceptor (target) membrane?

Answer 103

i. SNARES are transmembrane proteins that mark the identity of a particular vesicle. Example- v-SNAREs on trans-Golgi become incorporated into vesicles destined for synaptic vesicles. The target membrane (PM) has a different kind of transmembrane t- SNARE which marks it as the appropriate delivery (target) site. Interaction between v-SNARE and t-SNARE assures specificity of delivery. The mail carrier in this delivery system involves a number of additional proteins including Rabs and NSF.
ii. v-SNARE and Rab-GTP associate at donor membrane, bud off with clatharin. When proper target membrane is encountered (which has additional Rab-GTPs clustered on it), the v-SNARE and t- SNARE interact. Controlled cycles of GTP hydrolysis and GTP/GDP exchange control size and activity of the active Rab domains. The interaction of v- and t-SNAREs locks the vesicle onto the target membrane, allowing assembly of a fusion machinery. Membranes fuse. Fusion appears to be mediated by exposure of hydrophobic faces in proteins (fusogenic domains) which drive the lipids in the vesicles to intermingle with those in the target membrane. The vSNAREs and tSNAREs remain associated in the target membrane, and need to be pried apart by actions of NSF to recycle.

Question 104

Phagocytosis

Answer 104

Phagocytosis- specialized form of endocytosis in which large particles (e.g. cell debris, bacteria) are ingested (occurs in macrophages and neutrophils). Once inside the cell, the phagosome fuses with lysosomes, which release their contents and digest whatever was phagocytosed. Phagocytosis is triggered by specific recognition of target. e.g. antibodies on macrophage surface to bacteria will trigger binding and phagocytosis of the bacterium.
Not without error- some bacteria use phagocytosis to enter macrophages intact, they resist digestion, and then are circulated throughout body in immunologically protected intracellular environment of the macrophage.

Question 105

Pinocytosis

Answer 105

Pinocytosis- found in virtually all eukaryotic cells, which continuously ingest bits of their plasma membrane and extracellular fluid as clatharin coated vesicles. Mediates a relatively huge flux of membranes and water into cell, countering the exocytic flux of membranes coming from inside. Relatively non-specific.

Question 106

Endocytosis

Answer 106

Endocytosis. This process an efficient way of accumulating specific macromolecules from extracellular fluid. If receptor for molecule is on PM, binding of the molecule triggers the formation of a patch of receptors. This patch then forms a coated pit, then a coated vesicle. This allows concentration of specific macromolecules from extracellular fluid. Example: cholesterol is transported in blood as low-density lipoprotein complexes. These complexes are bound by specific receptors on body cells, which are then internalized into coated vesicles, the coated vesicles uncoat (forming endosomes) which, after fusion with lysosomal vesicles, form late endosomes and lysosomes. Once in the lysosome, the cholesterol is released and used for membrane synthesis. Humans with mutations in this pathway develop atherosclerosis prematurely and many die of heart disease.

i. This illustrates an additional point- many of these endosomes end up as lysosomes after fusion with hydrolytic enzyme vesicles and acidification of the vesicle by a vaculolar H+ ATPase pump. However, specific early endosome components can be removed and either recycled to the PM, or targeted elsewhere in cell. The LDL receptor is recycled to the PM after releasing its ligand, the LDL. In contrast, the EGF receptor can be degraded along with its ligand, EGF.
ii. Endocytosis is an important way cells have of downregulating cell surface proteins rapidly.

Question 107

Nucleus’ membrane

Answer 107

Nucleus is enveloped by two lipid bilayers (inner and outer). Inner membrane is lined with a protein layer (lamin). Outer membrane is studded with ribosomes and closely resembles ER (with which it communicates). Specialized nuclear pores pass through both membranes (perhaps 3000-4000 pores per nucleus).

Question 108

Where are proteins synthesized that are destined for the nuclear membrane

Answer 108

Proteins destined for the nuclear membrane (e.g. components of nuclear pores) are probably synthesized on membrane-associated ribosomes, analogous to ER membrane proteins.

Question 109

Proteins destined for nucleoplasm

Answer 109

Unlike all of proteins described until now, however, proteins destined for nucleoplasm are synthesized on cytoplasmic ribosomes and begin their journey as soluble cytoplasmic polypeptides.
1. Small proteins and other molecules (e.g. ). Some nuclear proteins are imported by binding indirectly to an import receptor through an separate adaptor protein.
3. Structure of nuclear pore complex: multiple proteins arrayed as a wheel around a central aqueous channel. Includes spoke-like structures and fibrils which protrude into both nuclear and cytoplasmic faces. Sort of like a button-hole joining inner and outer nuclear membranes.
4. When import receptor/protein complex encounters nuclear pore, it appears to associate with cytoplasmic fibrils containing FG repeats. Then hops from FG repeat to FG repeat to pore, allowing it to be transported across nuclear envelope in an energy- requiring process. Center of pore opens and closes like shutter of camera to allow big macromolecules through. Gated transport. Basic mechanics are understood. Nuclear targeted cargo associates with an import receptor in cytoplasm, passes through pore into nucleus. Ran-GTP in nucleus binds to import receptor and dissociates its cargo. Import receptor with Ran-GTP bound to it (but without cargo) translocates back to cytoplasm;. Ran-GTP in cytoplasm is then hydrolyzed to Ran-GDP, which releases from import receptor to allow import receptor to find a new cargo and begin the cycle again. Directionality comes from presence of a GTPase hydrolyzing protein in cytoplasm, GDP/GTP exchange protein (replaces Ran-GDP with Ran-GTP) in nucleus. Note that nuclear localization signal is not cleaved off, but remains on protein. Also note that in animal cells, the nuclear envelope dissociates during mitosis; after nuclei reform, all nuclear proteins have to be fetched back into new nuclei.
5. Pores work in both directions. mRNA, tRNA have to leave nucleus for cytoplasm (often in form of a ribonuclear protein complex). Ribosome proteins must go both ways: ribosomal proteins are synthesized in cytoplasm, enter nucleus, associate with rRNAs, form a pre-ribosome subunit, then exit back out nucleus into cytoplasm. Therefore, there are also signals (and adapters) for nuclear export distinct from those for nuclear import. Ran plays a reciprocal role in establishing directionality for the export rceptors: Ran-GTP promotes binding of targets to nuclear export receptors, which then pass out of the nuclear pore
complex into the cytoplasm.
Conversion of Ran-GTP into Ran-GDP in cytoplasm causes release of exported cargo. In this way, Ran-GTP/GDP gradient can control both nuclear import and export by affecting the binding of cargo to import receptors versus export receptors oppositely (Ran-GTP always high in nucleus; Ran-GDP high in cytoplasm). Import receptors and evolutionarily are structurally related to export receptors. Interestingly, a single nuclear pore can import one macromolecule while simultaneously exporting a different macromolecule, up to 500 time per second.

Question 110

Transport of proteins into mitochondria and chloroplasts (long answer…)

Answer 110

A. Mitochondria and chloroplasts are complex organelles with additional sub-compartments. e.g. mitochondria have internal matrix space and an intermembrane space, with an inner and outer membrane. Chloroplasts have the same two sub-compartments, plus a third (the thylakoid space, surrounded in turn by the thylakoid membrane).
B. Although both mitochondria and chloroplasts have their own DNA genomes and transcribe and translate some of their own proteins (most of which end up in the inner mitochondrial membrane and the thylakoid membrane), the vast majority of the chloroplast and mitochondrial proteins are actually encoded by the nucleus, and are translated on cytoplasmic polysomes.
C. Translocation into the mitochondrial matrix is mediated by a 20 to 80 long amino acid signal sequence (amphipathetic α-helix) which is recognized by mitochondrial membrane receptors, and which is cleaved off after entry of protein into mitochondria. This process requires energy, which is supplied both by the electrochemical proton gradient across the mitochondrial inner membrane and by hydrolysis of ATP
1. appears to occur in three steps- association of the protein with a receptor (TOM) in the outer mitochondrial membrane (requires ATP), followed by contact of the TOM complex with inner mitochondrial membrane receptor (TIM) and transport into matrix (requires proton gradient for translocation through TIM).
2. Proteins are imported in an unfolded state (threaded). Unfolded at cytoplasmic side by chaperone cytoplasmic Protein threads through TOM/TIM, then finally refolded in mitochondrial matrix by separate mitochondrial chaperone (hsp 70) requiring ATP; this refolding also helps drive the import of the polypeptide chain by pulling.
D. Targeting into the mitochondrial inner membrane and the intermembrane space requires two signals.
1. For some proteins, protein appears to be transported into matrix, N-terminal signal sequence is cleaved off, and a second signal peptide is exposed, resulting in insertion into inner membrane through the OXA complex transporter.
2. Alternatively, other proteins may directly initiate transfer into matrix, but have a stop transfer signal which interrupts process and causes release of protein as inner membrane polypeptide.
3. In either case, cleavage of the tether attaching the protein to the inner membrane would result in release of the polypeptide as a resident, soluble, intermembrane space protein.
4. Additional complexes acure for certain proteins. Proteins destined to become soluble β-barrels in the intermembrane space are imported into intermembrane space by TOM complex, then threaded back and forth to their final conformation in outer membrane by SAM complex. Similar multipass membrane proteins destined for inner membrane are first imported into intermembrane space by TOM complex, then inserted in inner membrane by TIM22 complex.
E. Transport of proteins into chloroplasts generally represents similar schemes as mitochondrial transport (though signals and details must be distinct, given that the same cell can have both types of organelles and cannot afford to get confused).
1. However, chloroplasts do have an extra membrane compartment, the thylakoid. Proteins targeted to thylakoids have an extra hydrophobic thylakoid targeting sequence.
2. Thus, thylakoid-destined protein first associates through N-terminal signal sequence with transporter on outer chloroplast membrane. Then, ATP hydrolysis drives protein through outer and inner membranes into stroma. The N-terminal signal sequence is cleaved off, exposing a second, thylakoid signaling sequence. The protein then associates with a thylakoid translocator, and is finally transported into thylakoid space (multiple pathways possible).

Question 111

What makes proteins targeting the thylakoid unique

Answer 111

chloroplasts do have an extra membrane compartment, the thylakoid. Proteins targeted to thylakoids have an extra hydrophobic thylakoid targeting sequence.

Question 112

How does the thylakoid-destined protein reach the thylakoid

Answer 112

thylakoid-destined protein first associates through N-terminal signal sequence with transporter on outer chloroplast membrane. Then, ATP hydrolysis drives protein through outer and inner membranes into stroma. The N-terminal signal sequence is cleaved off, exposing a second, thylakoid signaling sequence. The protein then associates with a thylakoid translocator, and is finally transported into thylakoid space (multiple pathways possible).

Question 113

How does translation into the mitochondrial matrix occur

Answer 113

Translocation into the mitochondrial matrix is mediated by a 20 to 80 long amino acid signal sequence (amphipathetic α-helix) which is recognized by mitochondrial membrane receptors, and which is cleaved off after entry of protein into mitochondria. This process requires energy, which is supplied both by the electrochemical proton gradient across the mitochondrial inner membrane and by hydrolysis of ATP

1. appears to occur in three steps- association of the protein with a receptor (TOM) in the outer mitochondrial membrane (requires ATP), followed by contact of the TOM complex with inner mitochondrial membrane receptor (TIM) and transport into matrix (requires proton gradient for translocation through TIM).
2. Proteins are imported in an unfolded state (threaded). Unfolded at cytoplasmic side by chaperone cytoplasmic Protein threads through TOM/TIM, then finally refolded in mitochondrial matrix by separate mitochondrial chaperone (hsp 70) requiring ATP; this refolding also helps drive the import of the polypeptide chain by pulling.

Question 114

What do membranes do besides delineate the cell and keep the inside in or the outside out

Answer 114

Membranes do much more than simply delineate the cell and keep the inside in or the outside out. They are also the main information interface for a cell. In single-cell microorganisms, information about surrounding environment (nutrients, physical conditions) is critical for proper response of cell.

Question 115

What role do membranes play in cell to cell interaction

Answer 115

Membranes also play critical role in the many cell to cell interactions that keep a multicellular organism functional. Unicellular life probably arose on earth 3.5 billion years ago, but multicellular organisms arose only 1 billion years ago- generated a need for elaborate signaling mechanisms for life in a cellular society.

Question 116

Can cell to cell signaling occur with unicellular organisms

Answer 116

Some cell-cell signaling, in fact, does occurs even at level of unicellular organisms. e.g. mating in yeast (e.g. a vs α factor, with appropriate receptors demarcating opposite mating types and preparing cells for mating; cAMP in aggregation of myxobacteria, quorum sensing in other bacteria). But cell to cell signaling became much more elaborate with advent of metazoans.

Question 117

Extracellular signals

Answer 117

The extracellular signal molecules may be freely soluble (such as are many hormones), or may be attached to the surface of adjacent cells or to an insoluble, extracellular matrix. Receptors in target cells mediate the recognition of extracellular signaling molecules. Many signaling molecules are impermeable, and cognate receptors must be accessible on plasma membrane of the target cell. Receptors that span membrane help transmit these extracellular signals to the inside of cell. However, some soluble signaling molecules are small enough or hydrophobic enough to penetrate lipid bilayers; receptors for these signals can therefore be intracellular (e.g. nuclear hormone receptors).

Question 118

Contact-dependent cell signaling

Answer 118

contact-dependent- immobilized product on surface of one cell interacts with receptor on adjacent cell

Question 119

Paracrine signaling

Answer 119

paracrine signaling (secreted product operates locally on adjacent cells)

Question 120

Synaptic signaling

Answer 120

synaptic signaling (secreted product operates quite locally, but signal for secretion may have initiated quite distantly). Target cell determined by proximity and by receptor specificity

Question 121

Endocrine signaling

Answer 121

endocrine signaling (secreted product, a hormone, is released into bloodstream and is distributed widely in organism). Target cell specificity determined in part by specificity of receptor expression.
Thus, both synaptic and hormonal signaling operate at a distance, but there are differences. Neurons transmit information very rapidly, hormones slower. Hormones
operate at very diffuse, low concentrations, neurotransmitters usually operate at locally high concentrations.

Question 122

Autocrine signaling

Answer 122

Actually, a fifth mechanism exists- autocrine (signaling molecule operates on the same cell type that secretes it). This is in essence a positive feedback loop which may assist in commitment of a particular cell type to a particular developmental decision, or may help to measure if certain critical mass of a particular cell type has been reached. e.g. light emitting bacteria or muscle mass in developing mammals.

Question 123

Gap junctions

Answer 123

Not truly extracellular signaling, but also relevant: gap junctions are specialized cell-cell contact points that permit small intracellular molecules to diffuse from cell to cell (e.g. cAMP, Ca++). These gap junctions also electrically couple the cells, permitting a variety of intracellular chemical and electrical signals to be shared.

Question 124

ACh actions and receptors

Answer 124

For example, acetylcholine stimulates skeletal muscle contraction, but inhibits cardiac muscle contraction. In this case, the two cell types have different kinds of acetylcholine receptors, but this isn’t the only basis of the difference: differences in response may be determined by differences in downstream signal transduction events.

Question 125

Three broad classes of cell surface receptors

Answer 125

Three broad classes of cell surface receptors- ion-channel linked, G-protein linked, and enzyme linked

Question 126

Ion-Channel linked signaling: voltage-gated channels (long answer…)

Answer 126

A. For many cells, electrical properties of the PM represent a critical signaling and information source.
1, Electrical aspects are an inherent consequence of having an ion-selective lipid bilayer. The membrane is a capacitor, consisting of an insulator (the lipid) surrounded on both sides by an electrically conductive fluid.
2. Leakage or active pumping of ions from one side of the membrane to the other, in the absence of associated counter-ions, generates an electrical potential (voltage). This membrane potential represents a source of potential energy.
3. Will first focus on how multicellular organisms have exploited this natural property as a means of transmitting signals with great specificity and with time courses on the order of milliseconds.
B. Membrane potential in animal cells is generated mainly by the Na+/K+ ATPase, which pumps three Na+ out for every two K+ pumped in.
1. No counter-ions are carried along, so this itself slowly tends to make the outside (+) and the inside (-): i.e. the Na+/K+ pump is electrogenic.
2. In addition, however, many membranes are more permeable to K+ than to Na+ (due to K+ “leak” channels), thus the leakage of K+ from inside to outside of the cell tends to make the inside even more negative and the outside even more positive.
3. Eventually, this flux stabilizes into an equilibrium, as the leakage of K+ ions out is counterbalanced by the developing voltage, which tends to drive the K+ back in. The situation at equilibrium can be predicted by the Nernst equation:
V= ln(Co/Ci) times a “constant” (the constant = RT/zF, where R is the gas constant, T is the absolute temperature, z is the charge of the ion, and F is Faraday’s constant).
For typical animal cells, V is between -20 and -200 mV.
C. Neurons are elongated cells specialized to exploit changes in this membrane potential for signaling rapidly over long distances (some neurons are over a meter long).
1. Typically consist of a cell body with a single long axon and several shorter, branching dendrites. Dendrites receive signals, axons transmit them. Due to branching, one nerve cell may receive as many as 100,000 inputs.
2. Like most animal cells, neurons are polarized with inside negatively charged, referred to as their resting potential.
3. Neurons carry signal down the axon as a wave of depolarization, referred to as an action potential
D. Mechanism of action potential
1. Neuronal PM contains voltage-gated channels for Na+. These channels are closed under normal membrane potential conditions, but open if the voltage across membrane drops. Thus, a transient drop in the membrane potential is self-proliferating, causes Na+ channels to open in adjacent patches of membrane, which in turn cause an adjacent drop in voltage, and so forth. In essence, a wave of depolarization sweeps down the axon (influx of Na+ can even transiently drives the membrane potential to +50 mV).
2. The Na+ channels are unstable in open position, soon close, and the K+ leakage channels fairly quickly restore the original (resting) negative membrane potential. In many nerve cells, the resting potential can be even more rapidly restored through the actions of additional voltage-gated delayed K+ channels, which open when the membrane potential drops, but more slowly (e.g. after the Na+ channels do so).
3. Note that a given nerve can carry quite a few action potentials without expending ATP, driven only by the pre-existing difference between inside and outside K+/Na+. The number of ions that actually cross the membrane per action potential is relatively small compared to the total number of ions present. However, eventually the ion gradient would be depleted in the absence of the Na+/K+ pump, which works to maintain it.
4. These action potentials can be recorded electrically by inserting an electrode in the neuron and measuring the depolarization.
5. Many peripheral and CNS nerves are surrounded by layers of myelin, a specialized lipid insulator laid down by Schwann cells and interrupted at Nodes of Ranvier. This myelin sheath permits action potentials to be transmitted at much more rapid rates than in unsheathed nerves by saltatory conduction (each depolarization “step” spreads passively further down the axon due to this insulation of the nerve sheath). In multiple sclerosis, these myelin heaths are destroyed, causing neurological problems.
E. Information about the voltage-gated ion channels
1. Each channel is either fully closed or fully open; when open the Na+ voltage-gated channel allows over 1000 Na+ ions to pass per millisecond. Single channels opening or closing can be seen by patch clamping.
2. Voltage-gated cation (Na+, K+, Ca++) channels are also found in muscle, egg and certain endocrine cells. Appear to be evolutionarily and functionally related. For K+ channel, each channel consists of four identical subunits, each having 6 membrane spanning α- helices and a antiparallel ß-sheet lining the inside of the channel. The ß-sheet determines the permeability characteristics of the channel when open (i.e. ion selectivity). At least for the K+ gated channels, an N-terminal domain on the cytoplasmic side appears to act like a plug which may occlude the channel under inactivation conditions. A series of (+) charged amino acids in one of the transmembrane domains may act to sense the voltage gradient and open the channel when the voltage falls.
3. Important to note that voltage gradient across thin bilayer is enormous (100,000 volts per cm) because membrane is so thin. Thus, changes in this voltage can easily result in changes in conformation of charged regions of channel, gating the channel open or closed.
F. Physiologically, action potentials travel from dendrites to cell body, then out again to end of axon. Yet, this is not an inherent property of the action potential itself (can artificially depolarize axon, and reverse wave of action potential). Directionality comes from chemical events at ends of nerves and muscles, and related to how action potentials jump from cell to cell

Question 127

What is an inherent consequence of having an ion-selective lipid bilayer?

Answer 127

For many cells, electrical properties of the PM represent a critical signaling and information source.
1, Electrical aspects are an inherent consequence of having an ion-selective lipid bilayer. The membrane is a capacitor, consisting of an insulator (the lipid) surrounded on both sides by an electrically conductive fluid.

Question 128

Membrane potential in animals is generated mainly by what?

Answer 128

Membrane potential in animal cells is generated mainly by the Na+/K+ ATPase, which pumps three Na+ out for every two K+ pumped in.
No counter-ions are carried along, so this itself slowly tends to make the outside (+) and the inside (-): i.e. the Na+/K+ pump is electrogenic.

Question 129

For typical animal cells, voltage is between what values

Answer 129

For typical animal cells, V is between -20 and -200 mV

Question 130

Neurons

Answer 130

Neurons are elongated cells specialized to exploit changes in this membrane potential for signaling rapidly over long distances (some neurons are over a meter long).

1. Typically consist of a cell body with a single long axon and several shorter, branching dendrites. Dendrites receive signals, axons transmit them. Due to branching, one nerve cell may receive as many as 100,000 inputs.
2. Like most animal cells, neurons are polarized with inside negatively charged, referred to as their resting potential.
3. Neurons carry signal down the axon as a wave of depolarization, referred to as an action potential

Question 131

Myelin sheath and Nodes of Ranvier

Answer 131

Many peripheral and CNS nerves are surrounded by layers of myelin, a specialized lipid insulator laid down by Schwann cells and interrupted at Nodes of Ranvier. This myelin sheath permits action potentials to be transmitted at much more rapid rates than in unsheathed nerves by saltatory conduction (each depolarization “step” spreads passively further down the axon due to this insulation of the nerve sheath). In multiple sclerosis, these myelin heaths are destroyed, causing neurological problems.

Question 132

Info about the voltage-gated ion channels

Answer 132

1. Each channel is either fully closed or fully open; when open the Na+ voltage-gated channel allows over 1000 Na+ ions to pass per millisecond. Single channels opening or closing can be seen by patch clamping.
2. Voltage-gated cation (Na+, K+, Ca++) channels are also found in muscle, egg and certain endocrine cells. Appear to be evolutionarily and functionally related. For K+ channel, each channel consists of four identical subunits, each having 6 membrane spanning α- helices and a antiparallel ß-sheet lining the inside of the channel. The ß-sheet determines the permeability characteristics of the channel when open (i.e. ion selectivity). At least for the K+ gated channels, an N-terminal domain on the cytoplasmic side appears to act like a plug which may occlude the channel under inactivation conditions. A series of (+) charged amino acids in one of the transmembrane domains may act to sense the voltage gradient and open the channel when the voltage falls.
3. Important to note that voltage gradient across thin bilayer is enormous (100,000 volts per cm) because membrane is so thin. Thus, changes in this voltage can easily result in changes in conformation of charged regions of channel, gating the channel open or closed.

Question 133

Ion-channels: the synapse and ligand-gated (transmitter-gated) channels (long answer…)

Answer 133

A. There are a few types of electrically active cells which are electrically coupled directly together, such that the action potential jumps from one to another (true in parts of heart). However, in most cases, transmission from one cell to another is by an intervening chemical signal- release of a neural transmitter. This occurs at specialized cell-cell contacts called synapses.
B. Arrival of action potential at pre-synaptic cell terminus results in a fusion of secretory vesicles containing the neural transmitter with the PM, thus releasing the neural transmitter into the extracellular space at the synapse.
C. At the post-synaptic cell, specialized ligand-gated channels bind the neural transmitter. In the case of an excitatory synapse, these channels then open, allow Na+ influx, and depolarize the post- synaptic cell membrane. If enough transmitter is released to open enough ligand-gated channels, the post-synaptic cell will depolarize enough to open the voltage gated channels and initiate a new action potential.
D. Synapses do not only transmit signal, but can modify it or process it.
1. Synapses can be stimulatory or inhibitory. e.g. ligand (transmitter)-gated channels can be permeable to Na+, which tends to depolarize the membrane, or can be permeable to Cl- or K+, which tend to hyperpolarize the membrane potential (Cl- concentration is typically higher outside than inside cell).
2. Synapses integrate all the signals impinging on nerve cell; whether or not nerve cell is depolarized enough to fire its own action potential depends on balance between excitatory and inhibitory stimuli. Each synapse induces a local gradient of de- or hyper- polarization. The combined total of all of these (influenced by distance) and the temporal summation of incoming synaptic signals on the cell body determines if action potential is released.
3. Examples of (usually) excitatory neural transmitters: acetylcholine, glutamine, serotonin.
4. Examples of inhibitory neurotransmitters: γ-aminobutyric acid and glycine
5. Some neurotransmitters bind to non-channel receptors and regulate nerve signaling indirectly- will describe later in course.
E. Detailed example- the neuromuscular junction, a transmitter-gated cation channel. 1. Best studied synapse
2. The neuromuscular junction is where the terminus of the axon spreads over a specialized region of the muscle cell. The axon terminus contains large numbers of secretory vesicles containing acetylcholine, a neural transmitter. The muscle cell membrane underneath has large quantities of densely packed acetylcholine receptors.
a. arrival of an action potential at the end of the axon induces fusion of the secretory vesicles with PM and release of acetylcholine into synapse. Appears to be related to an influx of calcium into the nerve ending by voltage-gated Ca++ channels, somehow provoking fusion of acetylcholine-containing vesicles with PM and release of acetylcholine into the synapse.
b. the acetylcholine receptor (on nerve cell) is composed of five transmembrane subunits (2 of one kind and 3 of another). The two identical subunits each can bind a molecule of acetylcholine, which induces a conformational change which opens the channel. The channel opens for 1 millisec, letting Na+ into the muscle cell, then closes again. This depolarizes the muscle cell enough to trigger the voltage-gated Na+ channels, which propagate the depolarization across the entire surface of the muscle cell.
c. Finally, this depolarization causes voltage-gated Ca++ channels in the muscle cell to open, ultimately releasing Ca++ from special internal storage compartments called the sarcoplasmic reticulum. The presence of free Ca++ in the cytoplasm of the muscle cell induces the actin/myosin complex to contract.
d. Synaptic transmission stopped by re-uptake of neurotransmitter by pre-synaptic cell (e.g. serotonin synapses), or in case of neuromuscular junction, by an enzyme (acetylcholinesterase) which destroys the transmitter.
e. Affected by drugs- Acetylcholinesterase inhibitors prevent synapse from resetting, keeping muscle depolarized and in contraction; used in many insecticides. Diisopropylfluorophosphate episode on campus. Bungarotoxin (from some snake venoms-e.g. cobras, such as recently escaped at the Bronx Zoo) prevents acetylcholine receptor channel from opening. Curare (used in surgery) blocks the channel.
F. Different transmitters and different synapses serve to process and transmit information within the CNS. Often there is substantial diversity in the manner in which alternative forms of subunit are expressed from different loci and/or alternative mRNA splicing to adapt the ion channel receptor to different needs in different cells. This signaling complexity is ultimately manifested at highest levels as "thought." Neural receptors important targets for pharmacological intervention. e.g. Prozac inhibits uptake of serotonin.
G. Adaptation is the process by which certain signals delivered in certain ways actually alter post- synaptic cell to make it harder to fire. One method: Ca++ activated K+ channel opens in response to raised Ca++ at cytoplasmic face of nerve cell membrane. Repeated depolarization over long period graduals increases Ca++ concentration in nerve through voltage-gated Ca++ channels. Rise in Ca++ opens Ca++ activated K+ channel, hyperpolarizing the membrane and making it more difficult to trigger new action potentials.
H. Conversely, long-term potentiation makes it easier for a post-synaptic neuron to fire if it already has been activated in specific ways in the past. Example: NMDA (N-methyl-D-aspartate) class of glutamate receptors only fire if glutamate present and the membrane already depolarized by AMPA glutamate receptors (depolarization in this fashion removes Mg++ from blocking the NMDA channel). NMDA receptors, in turn, are permeable to Ca++, accumulation of which internally triggers a cascade of changes making the post-synaptic cell more responsive to subsequent signals (e.g. by an increase in number of AMPA receptors). Important role in learning.

Question 134

Synapses

Answer 134

Synapses do not only transmit signal, but can modify it or process it.

1. Synapses can be stimulatory or inhibitory. e.g. ligand (transmitter)-gated channels can be permeable to Na+, which tends to depolarize the membrane, or can be permeable to Cl- or K+, which tend to hyperpolarize the membrane potential (Cl- concentration is typically higher outside than inside cell).
2. Synapses integrate all the signals impinging on nerve cell; whether or not nerve cell is depolarized enough to fire its own action potential depends on balance between excitatory and inhibitory stimuli. Each synapse induces a local gradient of de- or hyper- polarization. The combined total of all of these (influenced by distance) and the temporal summation of incoming synaptic signals on the cell body determines if action potential is released.
3. Examples of (usually) excitatory neural transmitters: acetylcholine, glutamine, serotonin.
4. Examples of inhibitory neurotransmitters: γ-aminobutyric acid and glycine
5. Some neurotransmitters bind to non-channel receptors and regulate nerve signaling indirectly

Question 135

Examples of how Ion fluxes and membrane potentials play important signal transmission roles in non- neuronal/non-muscular cell types.

Answer 135

Examples:
A. Some endocrine cells control their release of hormones in response to electrical signals.
B. In fertilization, entry of first sperm into egg cytoplasm triggers a rapid depolarization of the resting potential of the egg. This depolarization serves as the initial block to polypspermy, rapidly making the fertilized oocyte unable to fuse with additional sperm

Question 136

Cell-cell interactions mediated by non-electrical, G-protein linked receptors (long answer)

Answer 136

A. Represent the largest family of cell surface receptors.
1. Identified first by classical biochemical purification. More recently- additional family members have been identified by cross-hybridization with probes made from known receptors, or by expression cloning.
2. G-protein linked receptors respond to broad variety of signals, including hormones and neurotransmitters. Are among the most abundant cell surface receptors. Nearly a thousand different G-protein linked receptors in humans. Half of all known drugs work through G-protein linked receptors.
a. Despite a wide diversity in the nature of the ligands (ranging from proteins and small peptides, to single amino acids and fatty acid derivatives), the G-protein linked receptors themselves all share significant sequence relatedness. Evolutionary related?
b. They consist of a single polypeptide chain which crosses PM seven times- also known as “serpentine receptors”.
c. Extremely conserved in evolution (arose from single primordial gene?): indeed, the α and a mating factor receptors in yeast are also members of this same family.
d. In fact, the same general structure is also found in rhodopsin, which transduces light signals in vertebrate eye, and in olfactory receptors in nose.
B. The extracellular domains bind to ligand and initiate the signal. This signal is transduced across the membrane, where it is coupled to the trimeric-G proteins.
C. Trimeric G proteins act as molecular switches that flip between two states- active (when bound to GTP) and inactive (when bound to GDP). Remember Rab and Ran: are related switches.
1. Intriguingly, these same proteins also act as GTPases- in essence, these are molecular timing switches which self-terminate their own signaling.
2. Step-by-step mechanism:
a. The trimeric G proteins are flipped into their active state by interaction with a ligand- receptor complex (probably by inducing binding of GTP, leading to activation),
b. they then transmit the signal to a downstream target molecule.
c. Subsequently the GTP is hydrolyzed and the G-protein switches itself off and resets. GTP hydrolysis is stimulated by interaction with “GAP” or “RGS (regulator of G protein)” proteins.
d. Ultimately, the inactive G protein/GDP complex receives a new upstream signal, which induces replacement of inactive GDP with active GTP and repeat of the cycle. Replacement of GDP with GTP is stimulated by interaction with “GEFs” (GTP/GDP exchange factors).
3. Thus, GTP not used as source of phosphates or high energy, but more as internal clock.
4. The trimeric G-proteins then typically act downstream by creating an intracellular second messenger, which in turn passes on the signal to still other effector molecules.
5. Examples of these intracellular secondary messengers include cyclic AMP and Ca++ Both stimulatory (Gs) and inhibitory (GI)-protein complexes are known.

Question 137

Half of all known drugs work through what kind of receptor

Answer 137

Half of all known drugs work through G-protein linked receptors.

Question 138

Mechanism of Trimeric G-proteins acting as molecular switches

Answer 138

Step-by-step mechanism:

a. The trimeric G proteins are flipped into their active state by interaction with a ligand- receptor complex (probably by inducing binding of GTP, leading to activation),
b. they then transmit the signal to a downstream target molecule.
c. Subsequently the GTP is hydrolyzed and the G-protein switches itself off and resets. GTP hydrolysis is stimulated by interaction with “GAP” or “RGS (regulator of G protein)” proteins.
d. Ultimately, the inactive G protein/GDP complex receives a new upstream signal, which induces replacement of inactive GDP with active GTP and repeat of the cycle. Replacement of GDP with GTP is stimulated by interaction with “GEFs” (GTP/GDP exchange factors).

Question 139

How do eukaryotes maintain low cytoplasmic Calcium levels

Answer 139

To maintain low cytoplasmic Ca++, all eukaryotic cells have an ATP-driven Ca++ pump in PM. Muscle and nerve cells also have a Ca++/Na+ antiporter in PM to restore equilibrium after Ca++ influx caused by repeated stimulation, and a Ca++ pump in the ER to sequester Ca++ intracellularly.

Question 140

What are some Ca++ responsive proteins in cytoplasm?

Answer 140

a. troponin C in skeletal muscle, which regulates the actin/myosin contractile process
b. calmodulin-very abundant, widespread in all eukaryotic cells and functions as a multipurpose intracellular Ca++ receptor. Has four binding sites for Ca++ and undergoes conformational change when binding Ca++. The Ca++/calmodulin complex then regulates the activities of a broad assortment of different target proteins (some of which exist pre-bound to calmodulin, some of which only bind to calmodulin after Ca++ binding). Important category is the Ca++/calmodulin-dependent protein kinases (CaM-kinases). These include CaM-kinase II, which phosphorylates other proteins on serine or threonine residues. Intriguingly, CaM kinase II also phosphorylates itself, allowing it to stay active after Ca++ levels decline. A form of molecular memory and contributes to cognitive memory in mice.

Question 141

Binding of GTP-αs activates adenylyl cyclase, causing what?

Answer 141

Binding of GTP-αs activates adenylyl cyclase, causing ATP to convert to cAMP

Question 142

What is Calcium in relation to G Protein receptors?

Answer 142

Calcium as an intracellular signal regulated by G-protein coupled receptors