Flashcards in Exam 3 - Andreas slides READING checklist Deck (106):
What is live-cell imaging?
Allows us to directly observe molecular processes in living cells.
- Involves fluorescently labeled proteins to be injected and observed.
What can fluorescence microscopy reveal?
Location within a cell of a protein present in very low concentration.
- Used on antibodies that bind with high affinity to the protein being sought.
What is the function of in vitro motility assays?
Help detect the activity of an individual protein molecule acting as a molecular motor in "real time."
What is FRAP?
Fluorescence recovery after photobleaching
- Helps measure polymer dynamics.
Microtubules are components of what 3 things?
1. Mitotic spindle of dividing cells
2. Core of cilia
3. Core of flagella
What is the wall of MTs made of?
Globular proteins arranged in longitudinal rows called protofilaments.
- MTs consists of 13 protofilaments aligned side by side in a circular pattern within the wall.
What kind of interactions occur between adjacent protofilaments and what is the purpose?
Noncovalent interactions play an important role in maintaining MT structure?
The plus end of a MT is terminated by what?
The minus end of a MT is terminated by what?
What is tau?
A MAP that when expressed at abnormally high levels of phosphorylation lead to development of several fatal neurodegenerative disorders (EG. Alzheimer's).
How are MAPs controlled?
Primarily by the addition and removal of phosphate groups from particular amino acid residues.
MTs role in plant cells
Indirect role in maintaining cell shape through their influence on the formation of the cell wall.
MT roles in cells
1. Mechanical support
2. Provides shape of cells
3. Maintaining the internal organization of cells
What does axonal transport move?
1. Material (neurotransmitter molecules) are compartmentalized within membranous vesicles in the ER and Golgi complex of the cell body and then transported down the length of the axon.
2. Non-membrane bound cargo (RNAs, Ribosomes, cytoskeletal elements) are also transported down.
What is anterograde direction?
Structures and materials traveling from the cell body toward the terminals of a neuron.
What is retrograde direction?
Endocytic vesicles that form at the axon terminals and carry regulatory factors from target cells move from the synapse toward the cell body.
What classifies as the cytoskeleton?
2. Intermediate filaments
Kinesis and dyneins move along what?
Myosins move along what?
What are some things that motor proteins move?
1. Ribonucleoprotein particles
6. Other cytoskeletal filaments
What moves along intermediate filament tracks?
No motor protein known to do so.
What is different about intermediate filament tracks regarding polarity?
They are not polarized and thus would not provide directional cues to the motor proteins.
What two cycles do motor proteins employ to move along the tracks?
1. Mechanical cycle (conformational changes)
2. Chemical (catalytic) cycle: provides energy through ATP hydrolysis
What is KRP?
What is the responsibility of the globular heads of the kinesin molecule?
Binds to MT and acts as ATP-hydrolyzing, force-generating "engines."
The motor portions of KRPs have related amino acid sequences, why?
This reflects their common evolutionary ancestry and similar role in moving along MTs.
Why do the tails of KRPs have diverse sequences?
This reflects the variety of cargo that these motors haul.
What are differences between myosin and kinesin?
1. Kinesins are much smaller proteins.
2. Motors operate on different tracks.
Which direction does Kinesin travel?
Plus end-directed micro tubular motor.
EG. Cell body to synapse
The velocity of Kinesin is proportional to what?
The ATP concentration.
EG. Low ATP conc = slow
8 nm in length is equal to?
One tubulin dimer in a protofilament
What is processive?
Meaning that the motor protein tends to move along an individual MT for considerable distances without falling off.
Kinesins are usually well adapted for what kind of travel?
Independent, long-distance transport of small parcels of cargo.
What is different about kinesin-14?
Moves in the opposite direction (towards minus-end).
- Needs minus-minus neck regions to move this way.
- A plus-minus will always move in plus.
What is the Ncd protein of Drosophila?
A kinesin-14 protein that moves toward the minus end of the MT track.
What is different about kinesin-13?
Incapable of movement, aka MT depolymerases: bind to either end of a MT and bring about its depolymerization.
What is the protein responsible for the movement of cilia and flagella?
What is responsible for the MT-binding site on Dyneins?
The stalks that are found at the tip of the Dynein head.
Which direction does Dynein move?
Toward the polymer's minus end.
What are the two roles of cytoplasmic Dynein?
1. A force-generating agent in positioning the spindle and moving chromosomes during mitosis.
2. A minus end-directed MT motor with a role in positioning the centrosome and Golgi complex and moving organelles, vesicles, and particles through the cytoplasm.
What is the function of dynactin?
1. A multisubunit protein that acts as an adaptor for cytoplasmic dynein interactions with membrane-bounded cargo.
2. Regulates dynein activity
3. Helps bind the motor protein to MT = increase in processivity.
What sort of cargo are dynein-driven?
1. Endosomes, lysosomes,
2. ER-derived vesicles heading to Golgi-complex
3. RNA molecules
4. HIV virus
What does the function of a MT within a living cell depend on?
What two phases do MTs go through?
1. Nucleation (small portion of MT is initially formed)
2. Elongation (rapid phase)
What is MTOC?
Microtuble organizing center
What is the best studied MTOC?
Nucelation process in vitro vs in vivo
In vitro = slow
In vivo = rapid and associated with MTOCs.
What is the centrosome made of?
Two barrel-shaped centrioles surrounded by PCM (pericentriolar material = electron dense).
What are centrioles?
1. Cylindrical structure containing 9 evenly spaced fibrils, each of which has a band of 3 MTs (A,B, and C).
2. Always found in pairs.
3. Only A tubule is a complete MT and connected to center by a radial spoke.
What is the role of centrosomes?
Initiation and organization of the MT cytoskeleton.
- The site of MT nucleation
What is the role of PCM?
Initiates the formation of MTs.
What is the role of centrioles?
Recruits the surrounding PCM during assembly of the centrosome.
Where does elongation occur?
Elongation occurs at the plus end of the MT (opposite of the MTOC).
What is a basal body?
Another type of MTOC. Resides at the base of cilium or flagellum and generates MTs to the outer parts of cilia/flagellum.
Are basal bodies and centrioles interchangeable?
Pretty much. Identical in structure and can give rise to one another.
Do plant cells have centrosomes?
No, they don't have centrioles and any other obvious type of MTOC either.
Where are MTs nucleated in plant cells?
Around the surface of the nucleus and widely throughout the cortex.
What is the general role of all MTOCs?
1. Control # of MTs
2. Control polarity
3. # of protofilaments that make up their walls
4. Time and location of their assembly
What is commonly shared among all MTOCs?
Protein component known as gamma-tubulin (y-tubulin)
How common is gamma-tubulin (y-tubulin)?
Only about 0.005 percent of cell's total protein.
What is the function of y-tubulin?
1. Critical component in MT nucleation.
2. Determines the polarity of the entire MT and also forms a cap at its minus end preventing gain or loss of tubulin subunits.
What does injection of anti-y-tubulin antibodies into the living cell do?
Blocks the reassembly of MTs following their depolymerization by drugs or cold temperatures.
What is y-TuRC?
Gamma-tubulin ring complexes: found on PCM and are attachment sites for y-tubulin.
What are the four major arrays of MTs present during the cell cycle of a plant cell?
1. Distributed widely throughout the cortex.
2. Preprophase band that encircles the cell like a belt (marks site of the further division plane).
3. MTs reappear in the form of the mitotic spindle.
4. Spindle replaced by bundle of MTs called phragmoplast (plays role in formation of the cell wall that separates two daughter cells).
MTs that are less liable (less sensitive to disassembly):
MTs that are extremely liable (sensitive to disassembly):
1. MTs of mitotic spindle
2. MTs of cytoskeleton
How does GTP hydrolysis change conformation of MTs?
1. GTP is required for MT assembly.
2. A GTP molecule must be bound to beta-tubulin subunit for assembly of tubulin dimers.
3. Beta-tubulin is a GTPase that hydrolyzes GTP to GDP shortly after the dimer is incorporated into a MT.
4. GDP remains bound to assembled polymer.
5. When dimer is released from MT, GDP is replaced for GTP.
6. This nucleotide exchange "recharges" the dimer and allows it to serve as a building block for polyermization again.
How does GTP hydrolysis change the stability of MTs?
1. During rapid MT growth, tubulin dimers are added more rapidly than their GTP can be hydrolyzed.
2. Forces hydrolysis of GTP to GDP. GDP-capped tubulins want have a curved conformation = mechanical strain destabilizing the MT.
3. Strain energy is released as protofilaments curl outward from plus end and depolymerize rapidly shrinking the MT.
4. This is known as affecting the dynamic quality of MTs.
What is dynamic instability?
1. Growing and shrinking microtubules can coexist in the same region of a cell
2. a MT can switch back and forth unpredictably (stochastically) between growing and shortening phases.
- Plus end of MT = more dynamic
3. Allows MTs to quickly disassemble and be remodeled into things like the bipolar mitotic spindle.
What are +TIPs and their function?
1. External factors (protein) that binds to the dynamic plus ends of MTs.
2. Some regulate the rate of the MTs growth, shrinkage, or frequency of interconversion between two phases.
3. Others mediate the attachment of the plus end of the MT to a specific cellular structure.
Polymerization of a MT can _______ an attached object (MT plus +TIPs).
Depolymerization of a MT can ______ an attached object (MT plus +TIPs).
Pull on (major role in segregation of chromosomes during cell division).
MTs of Cilia and Flagella are stable or liable?
What are the characteristics of Cilia?
1. Moves in perpendicular direction.
2. Power stroke = cilium maintained in rigid state as it pushes surrounding medium.
3. Recovery stroke = cilium becomes flexible, offering little resistance to medium.
4. Occur in large numbers on a cell's surface.
5. Beating activity is usually coordinated.
What are the characteristics of Flagella?
1. Usually in singles or in pairs
2. Exhibits a variety of different patterns (waveforms).
What does cilia do in multicellular organisms?
Move fluid and particulate material through various tracts.
What do cilia do in humans?
The ciliated epithelium lining the respiratory tract peoples mucus and trapped debris away from the lungs.
What does a primary cilium do?
AKA nonmotile cilium: has a sensory function, monitoring the mechanical and chemical properties of extracellular fluids.
What regulates the pattern of the beat in the algal cell?
Internal calcium ion concentration.
What is the axoneme and its structure?
1. The core of the cilium.
2. Contains array of MTs that run longitudinally through the entire organelle.
3. In motile cilium/flagellum, contains 9 peripheral doublet MTs surround a central pair of single MTs. (9 + 2 array)
4. Plus ends at the tip of projection
5. Minus ends at the base.
6. Each peripheral doublet consists of one complete MT (A tubule) and one incomplete (B tubule = 10 or 11 subunits rather than full 13).
What is intraflagellar transport?
IFT is responsible for assembling and maintaing these organelles (Flagella).
What does Kinesin 2 do?
1. Moves complex arrays of IFT particles along protofilaments of the peripheral doublets to the assembly site at the tip of the growing axoneme.
How is kinesin 2 transported back and where?
Transported back to basal body by cytoplasmic dynein-powered mechanism (recycled axonemal proteins transported too).
The machinery for ciliary and flagellar locomotion resides _______ the axoneme.
What is ciliary (axonemal) dynein?
A protein that functions as an ATPase (hydrolyzes ATP).
What is the function of dynein arms?
The ATPase that releases the energy required for locomotion.
How do the dynein arms drive motility?
1. Dynein arms anchored along A tubule attach to binding sites on B tubule.
2. Dynein molecules undergo conformational change (power stroke) that causes lower doublet to slide toward the basal end.
3. Dynein arms detached from B tubule.
4. Arms reattached to upper doublet to that another cycle can begin.
What is glycocalyx and its function?
1. Mediate cell-cell and cell-substratum interactions.
2. Provide mechanical protection to cells.
3. Barrier to particles moving toward the llama membrane.
4. Binds important regulatory factors that act not he cell surface.
What is the ECM?
Extracellular Matrix: acts as a packing material or "glue" that holds cells together and provides both physical and biochemical signals that can play key regulatory roles in determining the shape and activities of the cell.
What is the basement membrane?
A type of ECM aka basal lamina.
1. Surrounds nerve fibers, muscles, and fat cells
2. Underlies basal surface of epithelial tissues (eg. epidermis of skin)
3. Underlies inner endothelial lining of blood vessels
(same roles as glycocalyx)
What are collagens?
Fibrous glycoproteins present ONLY in ECM.
1. Resistant to pulling forces
2. Single most abundant protein in the human body
What produces collagen?
1. Primary = fibroblasts
2. Smooth muscle cells
3. Epithelial cells
Two important structural features of collagen:
1. All collage are trimers consisting of three polypeptide chains called alpha chains.
2. Forms a rod-like triple helix
What is a proteoglycan?
Protein-polysaccharide complex: porous, hydrate gel that fills EC space like packing material and resists crushing (compression) forces. = compliments adjacent collagen molecules.
What makes up proteoglycans?
Chains of GAGs (glycosaminoglycans)
What are integrins?
1. Found only in animals
2. Integrates extracellular and intracellular environments
3. Binds to diverse array of ligands from extracellular environment.
4. Directly or indirectly interact with different proteins to influence the course of events within the cell.
What is the function of integrins?
1. Adhesion of cells to their substratum
2. Transmission of signals between the external environment and cell interior.
What are focal adhesions?
Specific sites where the cell is anchored to a surface.
- Plasma membrane contains large clusters of integrins.
- Acts as type of sensory structure (physical and chemical)
- Cell locomotion
- Consists of actin
What is a hemidesmosome?
A specialized adhesive structure found at basal surface of epithelial cells where cells are anchored to basement membrane.
- Consists of keratin (intermediate filaments: supportive function)
What is the IgSF?
- Mediates calcium-independent cell-cell adhesion (specifically for interactions of lymphocytes with cells required for an immune response)
What are IgSF VCAM?
Vascular Cell-adhesion molecule (member of IgSF)
- Mediation between non immune cells
What is IgSF NCAM?
Nueral cell-adhesion molecule (Member of IgSF)
- Mediation between non immune cells
What are cadherins?
Large family of glycoproteins that mediate Ca2+ dependent cell-cell adhesion and transmit signals from ECM to cytoplasm.
- Typically binds to other cahderins from neighboring cells.
What are adherens junctions?
Occur as belts (or zonulae adherents)
1. Cells held together by calcium-dependent linkages
2. Connect the external environment to actin cytoskeleton
3. Provide pathway for signals to be transmitted from cell exterior to cytoplasm
What are desomsomes?
Disk-shaped adhesive junctions found typically in tissues subjected to mechanical stress (cardiac muscle, epithelial layers of skin).
1. Contain cadherins