chapter 9,11, 12, 13

Question 1

how are antibodies used to visualize specific proteins within cells and tissues

Answer 1

• primary antibody binds to target protein (antigen A); secondary antibody (marker-coupled) recognizes primary antibody
• immunofluorescence; immunohistochemistry; western blotting

allows us to amplify signal because proteins can be very small. Multiple secondary antibodies can recognize a primary antibody to amplify signal

Question 2

What is fluorescence

Answer 2

a physical property of an object absorbing light at one wavelength and then reemitting it at another

Question 3

single molecule localization microscopy

Answer 3

• to determine the precise localization of specific molecules
• stimulated emission depletion microscopy

Question 4

what does EM tomography do?

Answer 4

• to study larger structures with same resolution as SEM
• specimen tilted to maximum of 60° to allow imagingn form multiple angles and give 3D reconstruction

Question 5

TEM sample prep

Answer 5

glutaraldehyde- fixative that cross-links proteins
osmium tetroxide- binds and stabilizes lipid bilayers and proteins
uranium and lead- staining tissue to visualize specific cell components

tissue stained with electron-dense materials, which adhere to the cells but dont target a specific protein

Question 6

cryo-EM

Answer 6

• specimen is rapidly frozen and imaged of all orientations without further processings
• can determine structure of macromoecules without need to crystalize

Question 7

What are 2 ways to color cells using light microscope? And what is it for?

Answer 7

1. Chromogenic dyes
2. Fluorescent dyes

to visualize cell compartments

Question 8

rank ease of passage of molecules through the membrane

Answer 8

1. hydrophobic molecules (gases, steroid hormones)
2. small uncharged polar molecules (H2O, urea, glycerol, NH3)
3. large uncharged polar molecules (glucose, sucrose)
4. ions

Question 9

channel vs transporter

Answer 9

channel- allow specific solutes to pass; faster transport rate; weaker binding to affinity to solutes; passive only
transporter- bind to solute and undergo conformational changes; may be passive or active (pumps)

Question 10

Sarcoplasmic reticulum

Answer 10

type of ER found in muscle cells

Question 11

active transport molecules

Answer 11

coupled transporter- using favorable gradient to drive unfavorable gradient
ATP-driven pump- ATP hydrolysis to power transport of something against its gradient
light-driven pump- light or redox energy to power transport against gradient (in bacteria, mitochondria, chloroplast)

Question 12

active or passive?
uniport
symport
antiport

Answer 12

both
uniport- moving one solute in either direction
symport- moving 2 solutes in same direction (coupled)
antiport- moving 2 solutes in different direction (exchange) (coupled)

Question 13

what type of transport is glucose transport from gut lumen to EC fluid?

Answer 13

passive simport because Na+ naturally wants to exit

Question 14

Vmax and Km

Answer 14

Vmax- rate at which transporter flips between conformational states
Km- affinitity to solute

Question 15

gating mechanisms for ion channels

Answer 15

• volatage-gated
• ligand-gated- extracellular ligand
• ligand gated- intracellular ligand
• mechanically gated

always passive

Question 16

How is an action potential initiated and terminated at a nerve terminus?

Answer 16

Initiated: voltage-gated calcium channels to open, allowing calcium ions to influx and trigger the release of neurotransmitters from synaptic vesicles
terminated :by the inactivation of sodium channels and the opening of potassium channels, which repolarizes

Question 17

• Identify the primary ions and channels involved in each phase of an action potential

Question 18

Role of inactivation in the action potential. What happens if channels do not inactivate?

Answer 18

Inactivation ensures that once an action potential has been initiated, the neuron cannot be continuously depolarized by the influx of Na⁺.

sustained influx of sodium ions, causing prolonged depolarization of the neuron. This could prevent the membrane potential from returning to its resting state.

Question 19

saltatory conduction

Answer 19

action potential propagates along myelinated axon jumping from node to node (just from one node of ranvier to another)

Question 20

What can action potential not cross?

Answer 20

a synapse

Question 21

schwann cells vs oligodendrocytes

Answer 21

schwan cells- myelinated axons in PNS
oligodendrocytes- myelinated axons in brain

Question 22

how can an action potential spread along distances?

Answer 22

by depolarizing neighboring regions of the membrane

Question 23

what controls direction of action potential propagation?

Answer 23

refractory period (Na channels are innactivated, so action potential is pushed to where they are closed)

Question 24

how can some molecules be at equilibrium, across a membrane but not be the same concentration on both sides?

Answer 24

electrochemical gradient

Question 25

nucleolus function

Answer 25

rRNA transcription and ribosome assembly

Question 26

stress granules functions

Answer 26

* temporary storage, particularly of translation-related components * formed in response to specific stress; contain mRNA

Question 27

p-granules functions

Answer 27

RNA metabolism and inheritance

Question 28

postsynaptic density function

Answer 28

organization of macromolecules in dendrites needed for neuronal transmission

Question 29

difference between a membrane bound organelle and a biomolecular condensate

Answer 29

**condensates**- membraneless organelles; formed from multiple weak interactions between proteins and macromolecules; subcellular organizations where proteins/nucleic acids are assembled into a functional unit; can form and dissolve PRN

Question 30

Explain liquid-liquid phase separation

Answer 30

* property of biomolecular condensate (membraneless organelles) * remains liquid but does not dissolve in the surrounding cytosol

Question 31

describe how the topology of intracellular compartments has evolved

Question 32

rough vs smooth ER functions

Answer 32

rough- translation of ER-destined proteins smooth- sites for budding and fusing of transport vesicles

Question 33

describe the mechanism that inserts proteins in the endoplasmic reticulum, for both soluble and transmembrane proteins, naming the key components

Question 34

what 2 kinds of proteins are transferred from cytosol to ER?

Answer 34

1. **soluble**: transferred completly to the Er lumen; destined for secretion out of cell or lumen of another organelle (not mitochondrion or plastid) 2. **transmembrane**: partly dislocated into ER, embedded in membrane; destined to reside in ER or membrane of another organelle or plasma membrane ## Footnote both initally directed to ER by ER signal sequence

Question 35

membrane-bound vs free ribosomes

Answer 35

**membrane-bound**: makes proteins that are being translocated to the ER **free**: make all other proteins

Question 36

ER signal sequence vs ER retention sequence

Answer 36

**signal**: N-terminal stretch of hydrophobic amino acids **retention**: C-terminus (KDEL amino acids)

Question 37

Explain how ER-designated proteins are recognized and transported

Question 38

Modifications that proteins undergo in the ER

Answer 38

**formation of disulfide bonds**- stabilize protein structure, only for proteins that are going to be secreted or have extracellular facing domain; oxidation of cystein **glycosylation** - carbohydrate layer for proteins that face extracellular side; aids with folding of proteins such as in case of calnexin

Question 39

Sec61 complex

Answer 39

translocator protein that inserts protein into ER

Question 40

What does accumulation of misfolded proteins in ER trigger?

Answer 40

UPR to make more ER to make more chaperones if misfolded proteins continue to accumulate and ER has reached full capacity, UPR directs cell death by apoptosis

Question 41

What happens if protein fails to fold in ER?

Answer 41

protein exported to cytosol and degraded by proteasomes

Question 42

Explain the molecular basis for cystic fibrosis

Answer 42

mutation in chloride transporter channel causes slight misfolding, which causes protein retention in the ER and degradation. Otherwise, the protein could be functional if reached the membrane disease is because channel cant fold it property

Question 43

Compare and contrast the transport to mitochondria and chloroplast

Answer 43

mitochondria primarily relying on an electrochemical proton gradient across their inner membrane while chloroplasts utilize ATP and GTP hydrolysis for import into their stroma and thylakoid lumen respectively

Question 44

What complexes move proteins across outer and inner mitochondrial membrane?

Answer 44

Outer: MIM1, TOM, SAM Inner: TIM22, TIM23, OXA

Question 45

how are mitochondrial proteins transported?

Answer 45

as unfolded polypeptide chains

Question 46

Name different compartments of mitochondria and chloroplasts and describe how proteins can be transported into each of the compartments

Question 47

Describe the nuclear pore

Answer 47

* "gates" in membranes of the nucleus that allow movement of molecules in both directions * small molecules can freely diffuse through nuclear pore but large molecules like proteins and RNA cannot pass without appropriate sorting signal/modificaitons

Question 48

Signal sequences and their function

Answer 48

* needed to pass through nuclear pore * short stretch of lysines/arganinines (positive charges)

Question 49

importins vs exportins

Answer 49

importins: nuclear import receptors; recognize nuclear localization signal and facilitate transfer of cargo into nucleus exportins: nuclear export receptors; facilitate transfer of cargo out of the nucleus

Question 50

Diagram the Ran GTP/GDP cycle

Question 51

How are nuclear import and export similar?

Answer 51

Ran GTP increases affinity to cargo when export changing the concentrations changes the direction of transport

Question 52

Compare and contrast the different transport systems between ER, mitochondria, chloroplast and nucleusx

Question 53

List the different secretory pathways and give example of secretory cells

Answer 53

COP2 coated vescicles: package and move collagen fibers (ER --> golgi) COP1: golgi --> ER clathrin: princh from plasma membrane

Question 54

Diagram protein movement in the Golgi

Answer 54

* Cis --> trans * proteins travel through cisternae by vesicular transportor by golgi cisternae themselves which migrate thourhg staacks * forward pathway: ER to golgi

Question 55

Explain the difference between phagocytosis and pinocytosis

Answer 55

phagocytosis: cellular eating; occurs in specialized cells that digest large molecules pinocytosis: ingestion of fluid and small molecules; all eukaryotic cells all of the time

Question 56

Explain receptor-mediated endocytosis, for instance of cholesterol

Answer 56

phosphorylation to turn on/off transport signals binding to anchoring proteins that localize compartments 1. cell bound to SREBP when dont need cholesterol. When found in environment because high cholesterol. 2. packaged into vesicle and sent to golgi 3. released transcription regulatory protein into cytoplasm 4. goes into nucleus and makes cholesterol biosynthesis enzymes

Question 57

Explain the difference between endosomes, late endosomes, and lysosomes

Answer 57

endosomes- large network of membrane tubes and vesicles that sort molecules early endosomes- just beneath plasma membrane; pH 7 late endosomes- closer to the nucleus; lower pH; involved in final stages of sorting and processing lysosomes- contain hydrolytic enzymes that digest macromolecules; lumen is acidic for enzymes

Question 58

autophagy vs mitophagy

Answer 58

**autophagy**- digestion of organelles by cell itself (signal: mTOR complex) **mitophagy**- selective destruction of mitochondria (PINK1 and parking promote ubiquinations)

Question 59

What is topologically equivalent to extracellular space?

Answer 59

ER lumen

Question 60

What organelle has similar topology to cytosol?

Answer 60

nucleus

Question 61

Long-term potentiation vs long term depression

Answer 61

**LTP**: strong stimulation of a postsynaptic neuron by a specific presynaptic neuron will generate reaction to subsequent weak stimulations, increase in synaptic length between 2 neurons **LTD**: result in loss of synapse between neurons

Question 62

Pathways that directly deliver materials to lysosomes

Answer 62

1. Endocytosis 2. Autophagy 3. Macropinocytosis 4. Phagocytosis