Chapter 9 Flashcards
How are microtubules arranged?
Hollow, cylindrical structures. They are a set of globular proteins arranged in longitudinal rows called protofilaments.
Microtubules contained _____ non-covalenty bonded protoflaments. Describe the structure of these
13.
Composed of dimers of alpha and beta tubulin subunits assembled into tubules with plus/minus ends
Why is there a plus and minus end of the microtubule?
Top molecules is beta tubulin (plus end) and bottom is alpha tubulin (minus end), alternating along microtubule, and because heterodimer structure is asymmetric the ends must be different.
What is the most significant difference between alpha and beta tubulin and why?
Beta tubulin has GDP in it whereas alpha tubulin has GTP. The GTP in alpha is not hydrolyzable, whereas the GDP in beta is
What does “MAPs” stand for? Why are they so important?
Microtubule-associated proteins. The structural integrity of microtubules is dependent on MAPs, which attach one domain to a microtubule while the other projects outward. They have been seen connecting microtubules to each other to maintian parallel alignment.
What do MAPs do and how are they regulated?
They attach to the surface of microtubules and increase stability and promote assembly. The binding activity of MAPs is regulated by phosphorylation of specific amino acid residues
_____ is a MAP protein found at Neurofibrillary Tangles in neurons. How do neurofibrillary tangles form?
Tau.
The tangles form when Tau protein is hyperphosphorylated (unable to bind to microtubules) and arranges itself in clumps - which happens in Alzheimers cases
How are microtubules related to axonal function?
Function in axonal transport, and axonal growth during embryogenesis
How do microtubules provide ultimate support to the plant cell? How are they arranged in relation to the long axis of the cell?
Help maintain cell shape by influencing formation of cell wall during interphase, influencing cellulose orientation (which is in the same places as microtubules). They are arranged perpendicularly to the long axis of the cell.
How do microtubules help with intracellular motility, especially in neurons?
A single axon can stretch much farther than the average cell (from spinal cord to finger for example). The cell body portion of the neuron is in the spinal cord and that is where proteins are produced. The axon is full of parallel microtubules for transport membrane bound cargo (vesicles, organelles) and non-membrane bound cargo (ribosomes, RNA), and mediate tracks for motor proteins.
What do motor proteins do?
Convert energy from ATP to mechanical energy. Move unidirectionally along their cytoskeletal track in a stepwise manner.
What are the three categories of molecular motors and what types of cytoskeletal elements do they move along?
Kinesin, dynein move along microtubules and myosin moves along microfilaments.
Describe the structure of kinesin-1
Kinesins are tetramers consisting of two identical heavy chains and two identical light chains. Each kinesin includes a pair of globular heads (bind to microtubule and act as HTP-hydrolyzing engine) connected to a neck, a rod-like stalk, and finally a tail that binds the cargo.
Describe the Dynein motor protein structure
Huge proteins composed of two heavy chains, as well as a variety of intermediate and light chains. Each heavy chain has a globular, force-generating head and a elongated stalk. Each stalk has a microtubule binding site. A longer projection (stem) comes off of the heads and connects to the intermediate and light chains
Requires an adapter (dynactin) to interact with membrane-bounded cargo.
Describe the Myosin motor proteins
Move along microfilament
Kinesins are members of a superfamily called ______. In which direction do they move?
Kinesin-related proteins. Anterograde, most move toward plus end of microtubule. Kinesin 13 does not move and Kinesin 14 moves toward the minus end of microtubule.
How would one prove experimentally that the only difference between kinesin 14 (which is retrograde) and the anterograde kinesins is in the head. What happened when this was done in the lab?
Swap the heads! Scientists did this in the lab and there was no change. They realized that it was actually the neck that caused the difference in movement
How do kinesins move?
Move along a single protofilament at a velocity proportional to ATP concentration. Protein moves a long distance without falling off.
Deynein is responsible for the movement of ____ and ____
Cilia, flagella
Distinguish between “DYNAMIN”, “DYNACTIN”, and “DYNEIN”
Dynamin - a collar protein found on vesicles
Dynactin - adapter protein
Dynein - motor protein (retrograde)
Microtubules running from ER to golgi are arranged in such a way that the ____ end is along the golgi complex
Minus
What are MTOCs?
Microtubule-organizing centers: Specialized structures for the nucleation/synthesis of microtubules
_____ microtubules are arranged in triplets around 1 centriole
9
______ are structures where outer microtubules (those in cilia and flagella) are created
Basal bodies
What controls the number of microtubules, their polarity, the number of proto filaments, and the time/location of assembly
MTOCs
1 dimer of tubulin, called _____ is very essential for other tubulins to grow and is found in all MTOCs. Why is this?
Gamma tubulin. They are thought to form to base for alpha and beta tubulin formation, as they are found in large concentrations in MTOCs but nowhere else
The base part of a microtubules, formed from gamma tubulin, is referred to as….?
Gamma-tubulin ring complex (gamma-TuRC)
How does gamma tubulin determine microtubule polarity?
Gamma tubulin forms the base of the microtubule, and the base end is minus end with a cap to prevent further dimers from being added. The plus end, then, must be the growing end.
What are the arrays of microtubules in a dividing plant cell
Interphase: distributed throughout cortex
Preprophase: single band
Metaphasd: mitotic spindle
Telophase: spindle disappears and forms phragmoplast
What are the two main ways that spatial organization of existing microtubules can be changed?
- rearrangement of existing microtubules
2. disassembly of existing microtubules and reassembly of new ones in different parts of the cell
How did in vitro studies of microtubule assembly influence our understanding of the process?
Helped us understand that GTP is necessary for microtubule formation (Beta-tubulin must be bound to GTP). It was found that GTP on beta-tubulin was quickly hydrolyzed to GDP
Describe the “Structural cap model of dynamic instability”
As microtubule is formed, two sheets close together through hydrolysis of GTP to GDP. GTP is present in the beta tubulin dimers are found at the head of the structure, but GTP hydrolysis is faster than dimer building, and GDP bound beta-tubulin does not easily stay in a straight structure, resulting in catastrophic breakdown of the microtubule.
How does the cell prevent the destabilization or the microtubule head?
MAPs will come along to stabilize the structure
How are MAPs activated or deactivated? What kind of enzymes do this?
Activated by phosphorylation via kinases
What is the difference between flagella and cilia in terms of structure and function?
Structurally similar with central “axoneme” consisting of microtubules in a 9+2 arrangement. Main difference between them is the way they move:
Cilia remain rigid during the power stroke and push against the surrounding medium, but become flexible during recovery stroke. Usually cilia are coordinated in movement.
Flagella are usually found in smaller quantities per cell and move in varying beating patterns to move cells in many directions.
What is a proto filament?
Structures that make up microtubules -13 of them per microtubules
How many microtubules are in a centriole and how are they arranged? How are they unique?
9 microtubules arranged in triplets. A microtubules are complete and attached to each other but B and C are incomplete (have less than 13 protofilaments)
In the axoneme, how many microtubules are there and how are they arranged?
9 microtubules in doublets, plus 2 in the middle
In both centrioles and axonemes, microtubules are attached to each other using _____
Nexin
In an axoneme, dynein molecules are attached to ______
A tubules
Flagella are assembled and maintained via _____. How does this occur?
Intraflagellar transport. Kinesin 2 moves complex intraflagellar transport particles and building materials along the peripheral doublet protofilaments to an assembly site on the tip of the axoneme. After this, recycled proteins and the kinesin are transported back to the basal body by cytoplasmic dynein
What machinery is required for movement of cilia and flagella?
Cilia dynein in the axoneme is required for ATP hydrolysis, supplying energy for locomotion
How does one break protozoan cilia for observing structures and ATPase activity?
- Treat with a detergent to break down cilia membrane. Centrifuge.
- Resulting pellet still has ATPase activity. Incubation with EDTA produces pellet of outer fibers (no arms) and supernatent with solubilized ATPase.
- Recombination in the presence of Mg2+ causes fibers to regain arms
Briefly describe the structure of microtubules
Microtubules - Cylindrical structures composed of alternating alpha and beta tubulin molecules on a gamma tubulin base.
_______ are a heterogenous group of proteins divided into five major classes. Which ones are found in the cytoplasm?
Intermediate filament (IF). Only I-IV are found in the cytoplasm
How is the study of Intermediate filament proteins helpful for many forms of research?
They are all found in different parts of the cell, and enable tagging and studying varying parts
How are IF proteins assembled?
Basic building block is a rod-like tetramer (formed of two antiparallel dimers with N and C-termini pointing in opposite directions). 8 tetramers associate with each other to form a filament, and these stack on top of each other in an end-to-end fashion to form a non-polar intermediate flament.
Describe the disease Epidermolysis bullosa simplex
Keratin (type 1) has a mutation and mechanical pressure results in severe blistering of skin
IFs containing _____ form the protective barrier of the skin, as well as epithelium of liver and pancreas
Keratin
What type of IFs are found in the neurons and how do they maintain proper spacing in the axon? What happens when they are damaged?
Neurofilaments (NFs), with sidearms branching off to maintain proper spacing between parallel neurofilaments of the axon. These provide support to the axon, which is large in diameter.
Aggregation of too many NFs can result in neurodegenerative disorders such as ALS and Parkinson’s disease.
What are microfilaments?
Composed of actin and involved in cell motility. Uses ATP to form flexible actin filaments (Called F-actin, actin filaments, or microfilaments), which have different structural characteristics and dynamic properties on each end.
What is the primary difference between microtubules and microfilaments?
Microtubules use GTP hydrolysis, whereas Microfilaments use ATP
_____ is the super family of motor proteins found in microfilaments
Myosin
How does one observe a microfilament orientation experimentally?
Treat it with the S1 head from myosin protein. This causes it to develop one pointed minus end and one barbed plus end. The orientation of the “arrowheads” allows one to observe the direction of microfilament movement.
Briefly describe the process of microfilament assembly/disassembly. What is it dependent on?
Dependent on concentration of actin monomers.
Leads to a drop in ATP-actin (ATP-actin is hydrolyzed to ADP-actin), and actin subunits are added to plus end and removed from the minus end. There must be equilibrium between assembly and disassembly.
How does the cell maintain an equilibrium between assembly and disassembly (called “treadmilling”)?
As Actin-ATP is used, concentration decreases and rate of addition eventually equals rate of removal - steady state with unchanging length.
_____ is the molecular motor of Actin filaments.
Myosin
What is the structure of myosin?
Has motor head for binding actin and hydrolyzing ATP, as well as divergent tail
What are the names of the two Myosin groups?
Conventional (type II) myosins and unconventional myosins
Describe the structure of myosin II and its function in the body
Two heavy chains connected to heads that wrap around each other to form a tail (which is needed to form filaments). The necks (leading to the heads) each have 2 light chains wrapped around them.
Myosin II are primary motors for muscle contraction but they also help split cell during division and aid in growth.
Describe unconventional myosins
They have a single head and cannot assemble filaments. Travel toward plus ends of the actin filament.
For example, myosin V is involved in organelle transport. Several of them are associated with cytoplasmic vesicles and organelles.
How does Myosin V walk along actin filaments
Walks like a math compass, using its long necks to swing heads, repeating itself (1 360 degree turn) every 36 nm
What will happen if there is a mutation in Myosin V?
Can cause partial albinism because melanophilin is carried to the surface and expresses itself there instead of being carried through the cell. Myosin-V works together with microtubules to deliver pigments around the cell.
Not all cases of albinism have Myosin V mutation. Why is this?
Sometimes it is a problem with pigment molecules themselves, or in Rab 27a (which attaches melanophilin to transport vesicles)
How are myosins related to hair cells of the ear?
triggering of stereocilia - which are arranged in a particular ordered fashion thanks to Myosin VIIa - results in opening of calcium gates, leading to a nerve impulse that results in hearing stuff.
Myosin VI is a reverse transported. Describe its action
Moves toward minus end of an actin filament. Involved in clathrin coated vesicles at the plasma membrane and transports uncoated vesicles to endosomes
Kinesins are all ____ end directed motor proteins, except _______
Plus, Kinesin 14
How are muscle fibers made in the embryo?
Fusion of myoblasts into one long multinucleate cell
Each muscle fiber contains hundreds of cylindrical strands called ______, banded with _____
Myofibrils banded with sarcomeres
Describe the structure of sarcomeres
Consist of banding pattern of thin (I band) filaments and thick (A and H band) filaments
“Thick” filaments in the muscle are composed of what? How are they placed in relation to thin filaments?
Hundreds of myosin molecules.
Each thick filament is surrounded by 6 thin filaments.
What happens to the muscle during contraction? What is actually happening during sarcomere contraction (which fibers are shortening?)?
“A” band remains constant in length. “H” and “I” bands decrease in width (until basically disappearing), and “Z” lines move inward. Once myosin comes in contact with actin filament, they bind and create “cross-bridges”.
During contraction, no fibers are shortening, but instead the thin filaments are sliding over the thick filaments.
What do the thin filaments contain and what purpose does each molecule serve?
Actin, tropomyosin and troponin.
Tropomyosin binds between two actin molecules
Troponin molecules are globular molecules that come in contact with both.
What purpose does “titin” serve in the sarcomere?
Provides structural support between thin and thick filaments, prevents muscle from being pulled apart.
What is the molecular basis for muscle contraction?
ATP hydrolysis induces conformational change in the head, and the neck acts as a swinging lever. The actin filament then slides a greater distance than would be possible without a lever
Describe, step by step and in detail, how myosin utilizes ATP to trigger muscle contraction.
- ATP binds to a cleft in the myosin filament, causing it to detach from the actin filament
- Hydrolysis of bound ATP causes the head to weakly bind to the filament
- Release of Pi causes tighter attachment of myosin head to the thin filament, and the power stroke occurs.
- Power stroke moves the thin filament closer to the M-line of the sarcomere.
The linking of the nerve impulse to the shortening of the sarcomere is referred to as ______
Excitation-contraction coupling
Action potential in muscles is propagated into the cell interior along _____. Where are these located?
Transverse tubules, which are located in close proximity to the cytoplasmic membranes of the sarcoplasmic reticulum.
What happens when an action potential is generated on the sarcomere?
Action potentials arrive by way of the transverse tubules. An action potential opens voltage gated calcium channels in the Sarcoplasmic reticulum, releasing Ca2+ into cytoplasm. The Ca2+ is bound to troponin, which causes a conformational change (allowing actin and myosin filaments to reach each other), shifting tropomyosin and exposing the myosin binding site, allowing myosin heads to bind to thin filaments.
What is the sarcoplasmic reticulum?
Smooth endoplasmic reticulum along muscle sarcomere with many many calcium ATPase pumps.
What are the three parts of the cytoskeleton? Define each.
Microtubules - Long, hollow unbranched tubes composed of tubulin protein
Microfilaments - Solid, thin structures organized into a branching network and composed of actin protein
Intermediate filaments - Tough, ropelike fibers composed of a variety of related proteins
How does the construction of the cytoskeleton lend itself to rapid assembly/disassembly
Each type of cytoskeleton filaments are held together by weak noncovalent bonds
What are the 5 major functions of the cytoskeleton?
- Providing structural support to determine shape of the cell and resist deformation
- Determining positions of organelles in the cell via internal framework
- Providing a network of “tracks” to direct movement of materials and organelles. Ex. delivery of mRNA, transport of vesicles
- Moving the cell from one place to another (ex. microtubules form cilia and flagella or movement in and outside of liquid)
- Essential for cell division (ex. microtubules form centrioles)
What is Live-cell imaging, what is involved and how did it contribute to our understanding of the cytoskeleton?
Live-cell imaging occurs when a fluorescence microscope is used to observe molecular processes in living cells with certain proteins tagged by GFP.
Either GFP labelled proteins are synthesized within the cell or protein subunits of cytoskeleton (ex. keratin or tubulin) are labeled with fluorescent dye and injected into a living cell to observe cytoskeletal movement.
How can fluorescence microscopy be used to reveal the location of a protein present in low concentrations in the cell?
Label an antibody for the protein and inject it into a living cell, OR add it to a fixed cell
What allows us to detect the activity of an individual protein molecule in real time? What has that allowed scientists to do in regard to the cytoskeleton, and how did they perform this experiment?
In Vitro single molecule motility assays. Observe, with fluorescence, the movement of a kinesin molecule down a microtubule in real time.
Microtubule was labelled with red fluorescent dye and kinesin with GFP.
How can atomic force microscopy supplement knowledge acquired through In vitro motility assays?
Can be used to measure mechanical properties of cytoskeletal elements, by doing things such as using the nanosized probe tip to test extensibility of filaments (by pulling them), which is how scientists found out about the extreme elasticity of intermediate filaments
How can one observe microtubule structure using FRAP (fluorescence recovery after photobleaching)?
Cell is either injected with GFP tagged tubulin or induced to produce it. A small section of the cell is bleached of fluorescence and the recovery of fluorescence signal in that area is a result of either microtubules being created in bleached zone or movement of microtubules THROUGH beached zone
How are microtubules generally arranged in cultured animal cells, giving them the characteristic round, flat structure? How does this contrast with columnar epithelial cells?
Radially from the nucleus.
Columnar epithelia animal cells have microtubules extended with their long axis parallel to the long axis of the cell, supporting the elongated shape.
How does treatment of a cell with colchicine (which promotes microtubule disassemby) influence organelles? What conclusion can be drawn from this?
It causes the golgi elements to disperse all over the cell, indicating that microtubules are needed to maintain the orientation of organelles in cytoplasmic space
Molecules moving from the neuronal synapse to the cell body of the neuron are moving in a _____ fashion
retrograde
Why is it not surprising that no motor proteins are associated with intermediate filaments?
They are not polarized and thus could provide no directional information
Motor proteins move cellular cargo such as….?
Ribonucleoproteins, vesicles, mitochondria, lysosomes, chromosomes, other cytoskeletal filaments
How do mechanical and chemical cycles work together to cause unidirectional motion of motor proteins/
Chemical cycle involves binding of ATP to the motor, hydrolysis of ATP, and release of ADP and Pi from the motor, followed by immediate binding of new ATP.
That chemical cycle drive the power stroke that moves the motor protein a set distance. This process repeats itself until there is either no energy left to fuel it or the motor protein reaches its destination
The ____ of all kinesin-related proteins have similar amino acid sequences, but the _____ have diverse sequences.
motor portions, tails
What is the currently accepted model for kinesin movement? Do kinesins fall off?
“hand-over-hand”, like walking on a tightrope. Two-headed kinesin molecules don’t tend to fall off because they are attached to both the microtubule and the cargo
How can one experimentally prove that kinesins are the primary motor proteins moving organelles in an anterograde fashion?
Create a mutant that has mutation in kinesin, then observe whether fluorescence tagged organelles have been distributed away from the cytoplasm
Distinguish between dynein and cytoplasmic dynein
Dynein is responsible for movement of cilia and flagella, whereas cytoplasmic dynein a huge protein that moves substances around the cell
Dynein motor proteins move in the ___ direction
Minus
Kinesin attaches to vesicle membranes by ____ and _____, whereas dynein attaches to vesicle membranes by _____
Peripheral and integral membrane proteins, whereas dynein attaches by the protein dynactin
What are the two primary functions of cytoplasmic dynein?
- Force generating agent in positioning the spindle nd moving chromosomes during mitosis
- As a minus end directed motor for positioning centrosome and golgi complex, and moving organelles, vesicles, and other particles
TRUE OR FALSE: A single vesicle or organelle cannot bind kinesin and dynein simultaneously
False, yes it can, enabling it to move in either a minus direction or a plus direction
What are microtubule organizing centers (MTOCs)?
Specialized structures that begin the nucleation (initial phase of microtubule construction) of microtubules. In animal cells, the primary MTOC is the centrosome
Describe the characteristics of the centrosome
Contains two barrel-shaped centrioles surrounded by amorphous pericentriolar material. Newly formed microtubules begin nucleation in the centrosome and radiate outward from it (not actually touching it, but instead residing in the pericentriolar material). in general the centrosome remains at the center of the microtubular network of animal cells.
Plant cells lack MTOCs. How are their microtubules arranged?
Microtubules of plant cells are nucleated around the surface of the nucleus and throughout the cortex
Which types of microtubules are the most stable? The least?
Cytoplasmic microtubules are extremely labile and sensitive to disassembly, whereas microtubules of centrioles, cilia, and flagella are very stable
What is meant by the term “dynamic instability” in reference to microtubule formation?
Microtubules are considered to be dynamic structures that switch between growing and rapidly shrinking in a matter of minutes. Microtubules can incorporate nearby alpha and beta tubulin into their structure and can switch from stability to instability quickly
How do +TIP proteins influence dynamic instability of microtubules? How does this relate to cell division?
+TIPs bind to the dynamic plus ends of microtubules, regulating rate of growth, shrinkage, and mediating attachment to kinetochore, actin cytoskeleton. Rate of growth/shrinkage in microtubules is the reason why chromosomes are pulled across the cell during cell division
Where does the ciliar/flagellar membrane come from?
Continuous with cell
Briefly describe the internal structure of the ciliary/flagellar axoneme
9 peripheral doublets of microtubules each consist of a complete A tubule and an incomplete B tubule. The central two microtubules are surrounded by a central sheath and connected to A microtubules by radial spokes. Each doublet is connected by an interdoublet bridge made of nexin protein. Two dynein arms are found on either side of the A tubules (outer and inner dynein arms)
If, after centrifugation in a lab, a pellet containing flagellar axoneme with missing outer dynein arms is created, would you expect the resultant flagellum to beat at all?
Yes, but only at half speed because it is missing half of the dynein arms that drive flagellar movement
In many cells ____ molecules form tiny bridges of elongated proteins between intermediate filaments
Plectins
IFs were originally thought to be permanent, unchanging structures. Why is that not thought to be the case any longer? What controls disassembly/assembly of IFs?
When labelled keratin is added to an in vivo cell, they are rapidly incorporated into the middle (not the ends) of existing IFs. This shows that they are as dynamic as other cytoskeletal elements. IFs assembly is controlled by phosphorylation of subunits
Which type of cell relies heavily on microfilaments?
Plant cells, which have restricted distribution of microtubules are forced to use primarily microfilaments to distribute vesicles and organelles
What is the role of Myosin I in the cell?
Not known…
Some vesicles contain microtubule based motors AND unconventional myosins. Why might this be?
At the end of a microtubule, a vesicle may have to switch to microfilament based transport to get to the desired location
The sarcoplasmic reticulum functions to store _____
Ca2+
