Structure and Function Exam 4

Kinesin vs myosin length of stride

Kinesin: 8nm myosin: 5nm

center point

MTOCs help to organize cells by establishing a center point - An isolated centriole mixed with free tubulin subunits seeks the center of an artificial "cell". (it moves from the corner of the artificial cell to the middle and by finding this central point it is adding organization to the whole scheme) - If we can replicate ourself then they are alive - A similar result is observed in a cellular system, fish pigment cells. (they took a piece of glass and pressed down on the cell in order to make a cell fragment which consists of microtubules that are severed form a new microtubule organizing center which are lacking centrioles

Microtubule growth

Reminder: Microtubule growth also depends on availability of more subunits than the critical concentration - hydrolysis almost always catches up and the GDP bound area is the minus end - We can have treadmilling, elongation, or shrinking

cohesins and condensins

Sister chromatids are held together by cohesins (so they dont get mixed up with the others and keep them together until everything is checked to make sure they will become daughter cells) and condensed into DNA coils by condensins (hinge 2 proteins an a clasp) • Cohesins and condensins have similar structures that allow them to bind both ATP and DNA

Microtubule Organizing Centers

The position and polarity of most microtubules is set by MTOCs (Microtubule Organizing Centers): • The minus ends of microtubules are bound by the MTOC, with the plus ends radiating out into the cytoplasm; this stabilizes and prevents depolymerization of the microtubule from the minus end. •The MTOC can be a centrosome (in most interphase cells), a basal body (in axonemes of cilia and flagella), or spindle poles.

Microtubule Structure

alpha a- and b-tubulin are distinct proteins that readily dimerize, and a/b dimer is basic building block of MTs • Both a - and b -tubulin bind GTP, but GTP hydrolysis only occurs on b -tubulin • Head-to-tail assembly of dimers gives linear protofilament • 13 protofilaments are arranged in a staggered helical array to form a hollow cylinder • MTs are polar: - exposed b subunits are at plus (+) end (fast-growing end) - exposed a subunits are at minus (-) end (slow-growing end) - they only stack/add to on one end (you cannot add to the middle) - 13 protofilaments need to be made in order to wrap around as a tube (cooperative forming because they are no longer adding as protofilaments but individual subunits.

Microtubule rings

are joined as laterally extended polymers - form a hollow tube called a singlet - some cilia and flagella form two hollow rings called a doublet (share a protofialemnt and there is an A ring and B ring) These rings are also connect to motor proteins that try to slide the two by each other but cannot and cause a twisting to occur (results in a whipping motion that leads to the movement) - centrioles and basal bodies tend to have triplet so there are 3 hollow rings (forms this large tube that looks like a centriole)

Rabbit oviduct

beating cilia propel eggs down the oviduct

g-TuRC

g-TuRC's appear to be critical to nucleating MTs

EB1

is a +TIP that helps to recruit other +TIP proteins including Catstrophin and XMAP215 - shown here tagged with green proteins and it can associate with GTP bound microtubules which tends to be the leading cap of microtubules and it does not stay bound to this side of the microtubule because it can surf around on the growing end - as we add new polymers, it is followed with depolymerization - EB1 stays associated with the polymerization - it acts as a connecting point two proteins

Kinesin vs myosin mechanism of movement

kinesin: "hand over hand" myosin: ratchet

Dynein-Driven Movement of Cilia & Flagella

• Cilia: numerous, hair-like. Whip-like beating causes movement of fluids, etc. over cell surface (or movement of single cells, e.g., Paramecium). • Flagella: longer, typically one per cell. Wave-like movement used to propel cell (e.g., sperm). (Note: bacterial flagella are made of flagellin protein and have different structures). • Both involve stable microtubules, in characteristic "9+2" arrangement, plus numerous associated proteins (dyneins, nexins, etc.) that make up the axoneme. • Basal bodies ("9 + 0") provide nucleation sites from which axonemal outgrowth occurs. • Ciliary dyneins have tails anchored in one microtubule doublet and heads in contact with a neighboring doublet. • Minus-end movement of dynein provides "sliding" forces that act on neighboring outer doublets; cross-links (due to nexin protein) translate force into a bending movement. • Coordinated movement requires appropriate spatial/temporal regulation of dyneins connecting different microtubule doublets.

Two things must occur during interphase for cell division to proceed

• DNA Synthesis - Each chromosome consists of two sister chromatids attached at centromeres and at many points along the arms by cohesin complexes. • In animal cells, centrosomes (containing centrioles) also replicate and will become the spindle poles. - New centrioles arise de novo. - Centrosome cycle: mitosis can be thought of as duplication and migration of centrosomes, during which time chromosomes are picked up and brought along into separate daughter cells.

Balancing catastrophes

• Enhance (catastrophins) or Suppress (XMAP215 - MAP protein similar to MAP 2 can bind to and stabilize microtubules but cannot coordinate bundles) - XMAP215 stabilizes • Phosphorylation of XMAP215 upon mitosis

General Overview of Microtubules (MTs)

• Largest diameter (~ 25 nm) of all three CSK elements. • "Hollow" rigid cylinders: stiffest of all three CSK elements. • Basic building block of microtubules is dimer of a and b tubulin. • In cells, MTs extend from "MTOCs" (microtubule *organizing centers*): e.g., centrosomes, spindle poles or basal bodies. (organizing centers associate with each other) • Some assemblages of MTs are "permanent", others are dynamic. MTs have the ability to rapidly disassemble in one location and reassemble in another. • Two kinds of motor proteins - dyneins and kinesins (minus end oriented)- direct movement along MTs .

Overview of Mitosis

• Mitosis equally partitions newly-replicated chromosomes into two daughter cells. • The mitotic apparatus is a specialized, constantly changing microtubule structure that is designed to capture, align and then separate the duplicated chromosomes. • In a rapidly dividing population of animal cells, mitosis takes ~1 hour. The cell is in interphase the rest of the time. • Mitosis is a continuous process, but is divided into 5 (or 4) phases: - Prophase - Prometaphase - Metaphase - Anaphase - Telophase *Review Panel 17-1*

Microtubule-Specific Motor Proteins

• Movement can occur in both directions along a single (polar) microtubule. • ATP hydrolysis drives movement along filament. • For a given motor, movement is unidirectional (kinesins and dyneins)

What is the role of GTP hydrolysis in microtubule assembly?

• Only b subunit has intrinsic GTPase activity • Plus ends of microtubules are stabilized when GTP-subunits are present (i.e., there is a "GTP cap"). • GTP hydrolysis causes a weakening of the non-covalent interactions between the GDP-tubulin subunits; this weakening destabilizes the + end of MT's and allows rapid depolymerization of the MT under certain conditions • What would be the effect of adding a non-hydrolyzable analogue of GTP to an in vitro assembly system of MTs?

Cytoplasmic dyneins

• transport vesicles along microtubules • are linked to membranes and vesicles through the dynactin complex (where we forma connection between microtubules and an actin like filament) - Arp 1 filament is forming a strong surface attaching site on the vesicle - without this attachment surface there is not much to hold onto

Basal bodies

("9+0" conformation) anchor the microtubules of the axoneme at the cell surface; similar to the way centrioles anchor cytoplasmic microtubules in the MTOC - 0 because there is nothing in the middle other than scaffolding proteins

Katanin

(Japanese for sword) • Severs MTs • Must slice across 13 bonded surfaces • Small subunit ATP dependent severing protein • Large subunit centrosome directing protein

MAP2 and Tau

- cross-link and stabilize microtubules - over expressing each of these proteins - when we over express MAP2 - cell elongates and takes on neuronal morphology - in tau (shorter arm) - we get dense parallel packing of the cell and this is present in diseases (we need lots of these to long arrays of microtubules in the neuron but susceptible to aggregation and unfolding)

loss of GTP - cap during microtubule assembly

- the protofilament that has been building rapidly has a GTP cap where as the when the cap is lost and depolymerization occurs - GDP must be replaced with GTP in order to have growing filaments

Ciliary dynein

Ciliary dynein binds doublet microtubules through both its tail and globular head regions, forming bridges between microtubules.

Kinesin vs myosin Processivity (# of cycles without falling off filament)

Kinesins: 100s of cycles Myosin: 1-2 cycles

MT binding Proteins

Microtubule-associated proteins (*MAPs*) link microtubules to one another and to other structures in cells. • MAP2 • Tau • EB1 • Catastrophin (Kinesin-13) • XMAP215 • Stathmin • Katanin

The microtubule monomer, is in fact, itself a hetero:

heterodimer

Three steps to assembly of MTs

(1) Assembly of short protofilaments --> (2) Sheet assembly to form a short microtubule with 13 protofilaments --> (3) Microtubule elongation by addition of dimers to the ends of protofilaments that make up the microtubule wall.

Microtubule Assembly

*Dynamics and Regulation* • Microtubules are highly dynamic in vivo: exchange between assembled subunits and (large) free pools in the cytosol. (similar to the critical concentration concept where the same number of subunits that are being added are also being removed) • Microtubule assembly is similar in many respects to that of actin microfilaments • In cells, minus ends are stabilized by anchoring in centrosome or other MTOC, so most instability (polymerization/depolymerization) occurs at + ends.

dynamic instability

*Microtubules show dynamic instability (oscillations between assembly (polymerization) and disassembly (depolymerization)):* • Not all microtubules behave identically under identical conditions • Occurs at concentrations near the Cc • Is due to (slow) polymerization when GTP-tubulin is present at the + end of MT's and (rapid) dissociation when GDP-tubulin is at the + end. • Dynamic instability can be regulated in cells (e.g., increased during mitosis and decreased in a differentiated state, such as in a cilium). There are rapid fluctuations

Stathmin

*can bind two MT dimers and decrease available subunit concentration* • Inactive when phosphorylated • Regulation of dynamic instability • Development of the neurons of the amygdala • Scared mice

Microtubules in cilia and flagella

*have a characteristic "9+2" arrangement (9 doublets around periphery, 2 single microtubules in center)* - The microtubule core of cilia and flagella is called an axoneme. - this is anchored in the basal body which is the organizing center which creates this microtubule array - microtubules are prevented from sliding past one another by linking proteins, and flagella bend instead (sperm cells require this for motility) --> can lead to infertility if this motor does not function well

Centrosomes

*set the MTOC in interphase cells* - are composed of a centrosomal matrix that contains 50 or more copies of g-TuRC (gamma tubulin ring complex), which nucleates and anchors microtubules. - Animals contain a pair of centrioles, an array of triplet microtubules. Centrioles do not make direct contact with MT - ends; MT's are nucleated by g-TuRC Centrosomes, the MTOC's in most interphase cells, contain centrioles and g-TuRC's in a centrosomal matrix. (sit in the middle of a gel organizing center) centrioles are made by microtubule triplet

Dyneins

*usually minus-end directed: mediate retrograde axonal transport, inward movement of Golgi, movement of cilia and flagella* - Kinesins (+ directed) and dyneins (- directed) are microtubule specific cargo-binding motor proteins • cytoplasmic dyneins (2-headed) are important for vesicle trafficking • ciliary or axonemal dyneins (2- or 3-headed) are important for movement of cilia and flagella - the MT-binding domain is also the motor domain

Kinesins

*usually plus-end directed (KIFC2, Ncd are exceptions): mediate anterograde axonal transport, outward movement of ER, chromosome segregation during mitosis/meiosis* - Kinesins (+ directed) and dyneins (- directed) are microtubule specific cargo-binding motor proteins - one specific kinesin gene can have an exception which can move towards the minus end (but this does not mean it can move towards the plus end) - share similar motor domains, but have unique structures that allow them to bind and transport different cargos

Kinesin vs Myosin movement activity

- Kinesin: they share 50% attached and detached the two heads work closely together - myosin: one motor work independently and one myosin is taking small powerful grabs against acting (in its cycle it si only attached for a short amount of time to create the power stroke

Functions of MTs

- Major determinant of cell polarity and cytoplasmic organization - Organelle movement along MTs - Axonal transport in neurons along MTs - Chromosome segregation during mitosis: the mitotic spindle - Movement of cilia and flagella: carried out by "permanent" assemblages of microtubules, with movement generated by MTs sliding over each other

Structure of microtubule motor proteins:

- Microtubule binding, ATP binding, ATPase activity and motive force is provided by globular head. Tails bind to "cargo." - Kinesins and dyneins are two headed (due to dimerization of heavy chains). Some ciliary dyneins are three headed.

• Microtubule assembly is similar in many respects to that of actin microfilaments

- Polymerization is cooperative - Polymerization occurs only at ends of MTs - Kinetics of microtubule assembly depends on critical concentration (Cc) of dimers in cytosol - Assembly and disassembly of MTs preferentially occurs at plus end.

What would be the effect of adding a non-hydrolyzable analogue of GTP to an in vitro assembly system of MTs?

- The GTP gamma s that is added is also known as a non-hydrolyzable analogue - you are going from interphase to prometaphase and the mitotic spindles are forming - having GTP gamma s would result in more stable microtubules thus making it much harder to take apart - we will stall and delay the mitosis process by making it harder to dissociate

Motor domains in Kinesins

- causes movement in a single direction - the dark green foot (lagging head) is moved towards the front to become the leading head - the lagging head is bound to ATP and microtubule surface - the leading head is bound to ADP and tightly bound - hydrolysis of ATP in the lagging head making it not tightly bound - there is communication between the lagging and leading strand and ATP going in causes a conformational change causing the leading head to be more tightly bound thus becoming the lagging head - the lagging head gets let go when the ATP bound leading head it swings towards the front (because the neck of the leading head goes through a strong conformational change)

Overview of cell cycle

- everything that the cell needs to survive needs to be passed on - this is done through the division of the cell

Mechanism of action for kinesin

- two heads step in processive fashion along the microtubule - spends more time in contact with microtubule than myosin does with actin

Changes in the GTP capping of + ends lead to dynamic instability

- when the GTP caps are lost then we have rapid shrinking because the GDP caps are exposed - when we depolymerize rapidly then we have a cloud of subunits available and the subunits can get recharged (leads to the rescue)

stabilizing GTP-cap

A stabilizing GTP-cap forms during rapid microtubule assembly: GTPbound subunits are linear and more stable. - this linear subunit would be called a protofilament - had to be in the gtp state in order to use (when there is gdp it is more likely to be depolymerized -- thus making it more dynamic) - when more subunits are added the conformational change causes the GTP to turn into GDP and creates a little curve - since the minus end is associated with the organizing center, so are less focused on this - rapid polymerization and depolymerization at the + ends and the minus ends are anchored to different structures

Which of the following is true of both actin microfilaments and microtubules? A. Hydrolysis of nucleotides leads to destabilization of both filaments B. Both are assembled from dimers present in the cytosol C. Both bind GTP D. Both radiate from an organizing center in the cytosol

A. Hydrolysis of nucleotides leads to destabilization of both filaments *exam style question*

Dismantling MTS

A. Stathmin can bind two MT dimers and decrease available subunit concentration B. Katanin