Cell Organization and Movement, Part 2: Microtubules and Intermediate Filaments

Lecture 21

aB-Tubulin assembles into dynamically

unstable and polarized microtubules with (+) and (-) ends and 13, 13+10 and 13+10+10 protofilaments walls

assembled tubin hydrolyzes

GTP

MAPs mediate the

assemble, dynamics, and function of microtubules

all microtubules are nucleated from

microtubule-organizing centers (MTOCs), and many remain anchored by their (-) ends 

centrosome MTOCs consist of

two centrioles and the surrounding pericentriolar material

the cytoskeleton has many roles:

• serves as a scaffold providing structural support and maintaining cell shape

• serves as an internal framework to organize organelles within the cell

• directs cellular locomotion and the movement of materials within the cell 

microtubules strcuture and composition 

• hollow, cylindrical structures

• set of globular proteins arranged in longitudinal rows Called protofilaments

• contain 23 protofilaments

• each protofilament is assembled from dimers of a- and B-tubulin subunits assembled into tubules with plus and minus ends

tubular dimer

• composed to stabely associated, highly conserved (in eukaryote), and structurally similar a-tubulin and b-tubulin monomers 

• most eukaryotes have several genes encoding both dimers and additional genes encoding a gamma-tubulin subunit, which is involved in MT assembly 

a-tubulin - GTP is

never hydrolyzed and nonexchangeable

B-tubulin - GDP is

exchangeable with GTP, which can be hydrolyzed in the site

tubular subunit organization in a microtubule

forms a structurally polarized tube

tubular dimers are aligned

end-to-end in the same orientation into protofilaments

tubular dimers- protofilaments pack

side-to-side with the same subunit polarity to form the wall of the microtubule

tubular dimers - protofilaments are slightly staggered so that

a-tubulin in one protofilament is in contact with a-tubulin in the neighboring protofilaments, except at the seam, where an a-subunit contacts a B-tubulin 

dimer alignments provides

structural polarity to the MT 

tubular dimers - subunits are added perferentially at the

(+) end where B-tubulin monomers are exposed

singlet microtubule:

13 protofilaments - most cytoplasmic MTs

doublet microtubules:

an additional wall of 10 protofilaments from a second tubule in cilia/flagella outer doublets

triplet microtubules:

two 10-protofilament walls on the 13-microtubule organizing centers 

microtubules are assembled from MTOCs to

generate diverse configurations

MTOCs-

specialized structures for the nucleation of microtubules

centrosome

responsible for iniatating microtubules in animal cells

centrosome structure

contains barrel-shaped centrioles surrounded by perientriolar material (PCM)

plants do not have 

centrosomes and basal bodies, but use other mechanisms to nucleate the assembly of microtubules

microtubules grow out from the

PCM

microtubule nucleation: role of

y-tubulin in centrsome function

MTOCs control the

number of microtubules, their polarity, the number of protofilaments, and the time and location of their assembly

the protein y-tubulin is found in all

MTOCs and is critical for MT nucleation 

individual mirctotubule (+) exhibits

dynamic instability with alternating period of growth and rapid disassembly (catastrophe), depending on GTP-cap or GDP-cap status

assembling microtubules store energy derived from GTP HYSROLYSIS IN

the microtubule lattice and can do work when disassembling

dynamically unstable microtubules can

“search” the cytoplasm and “capture” targeted structures or organelles

microtubule are dynamic structures that can

assemble or disassemble rapidly at both ends

growth or shrinkage of microtubule controlled by 

subunits at end of microtubule 

tubulin has GTPase activity

• can hydrolyzes GTP to GDP

• a-tubulin: bound to GTP, NOT hydrolyzed

• b-tubulin: bound GTP, can be hydrolyzed to GDP

catastrophe - 

microtubule shrinks very fast 

GTP-B-tubulin cap:

•MT with GTP-β-tubulin on the end of each protofilament

•Lateral protofilament-protofilament interactions in the GTP-β-tubulin cap are sufficiently strong to prevent protofilament unpeeling at the MT end.

•Strongly favors assembly by adding more GTP-tubulins.

•Microtubules grow.

GDP-B-tubulin cap 

•MT with GDP-β-tubulin at the end of each protofilament

•Protofilaments curve and undergo rapid disassembly.

•Disassembled GDP-β-tubulin exchanges GTP for GDP to become GTP-β-tubulin.

switch from assembly to disassembly(catastrophe):

rate of GTP hydrolysis (constant) is greater than rate of GTP-tubulin addition

switch from disassembly to assembly (rescue):

rate of GTP-tubulin addition is greater than rate of GTP hydrolysis

GTP-B-tubulin “islands” can persist

along the length of an assembled microtubule

• when disassembling mictotubules encounters a GTP-B-tubulin island, disassembly pauses and rescue may be initiated 

individual microtubules exhibit dynamic instability -

rapid microtubule polymerization alternate with periods of shrinkage

microtubule lengths plotted over time exhibit dynamic instability:

•Assembly and disassembly each proceed at uniform rates, but disassembly is much more rapid (7 μm/min) than assembly (1 μm/min).

MT (+) ends make abrupt transitions from elongation to shrinking (catastrophe) back to elongation (rescue)

side-binding MAPs stabilizes

microtubules

(+) end-binding +TIPs can alter microtubule dynamic

properties or attach cell components to the (+) end

microtubules ends are destabilized by proteins such as

the kinesin-13 family of proteins and Op18/stathmin, which enhance catastrophe frequency

Microtubule-associated proteins (MAPs)

• comprise hetergenous group of proteins

• attach to the surface of microtubules increase their stability and promote their assembly

MAP long arm and short arm

MApP2 (l) and tau (s)

MAP-MT associations

•Spacing reflects difference between the lengths of the MAP2 and tau projection domains.

•Side associations with several monomers along protofilaments stabilize MTs and dampen dynamic instability.

•MAP/tau phosphorylation can regulate MT interactions.

proteins that destabilize ends of MTs → kinesin 13:

•Enhances the disassembly of either a (+)/(–)-MT end

•ATPase activity dissociates Kinesin-13 from the αβ-tubulin dimer.

proteins that destabilize ends of MTs → Op18/stathmin binds 

selectively to two dimers in curved proflaments and enhances their dissociation from a MT end

• activity is inhibited by phosphorlyation at a cell’s leading edge, contributing to MT growth at from of cell 

the kinesis (+) end motor superfamily transports

organelles and slides antiparallel microtubules past each other

kinesin-1 is a higher processive motion because it

coordinates ATP hydrolysis by its two heads so that one head is always firmly bound to a microtubule

cytoplasmic dyne is a (-) end motor that associated with

the dynactin complex and cargo adapters to transport cargo

tubular post-translational modifications stabilize

microtubules and regulate activity to interact with motors

kinesin-1 powers

vesicle movement down axons toward the (+) ends of microtubules

kinesin-1: homonymer of two identical heavy chains

•Head motor domain – MT and ATP/ADP binding sites

•Flexible linker domain – required for motor activity and connects head to the coiled-coil stalk

Two light chains associated with the tail of each heavy chain bind to receptors on vesicles

kinesis-head domains: x-ray structure

•Microtubule-binding site

•Nucleotide-binding sites (containing ADP)

•Linker regions connect heads to stalks.

model of kinesin-1-catalyzed vesicle transport 

•Attaches to a vesicle surface receptor

•Transports vesicles from the (−) end to the (+) end of a stationary microtubule

•Movement is processive, motor protein moves along an individual microtubule for a long distance without falling off.

kinesin superfamily - kinesin-1

involved in organelle transport

kinesin superfamily - kinesin-2

family has two closely related but nonidentical heavy chains and a third cargo-binding subunit 

kinesin superfamily - kinesin-5

four heavy chains assembled in a bipolar configuration can slide antiparallel microtubules pas each other 

kinesin superfamily - kinesin-13`

• “motor” domain in the middle of heavy chain has no motor activity 

• destabilize microtubules ends for disassembly 

kinesin cycle: processive

one head is always bound to the MT - can take thousands of step without dissociating from MT

kinesin cycle start

•Leading head with no nucleotide bound is tightly associated with the microtubule.

•Trailing head with ADP bound is weakly associated with the microtubule

kinesin-1 inhibited form

• head back and interacts with tail 

• inhibits the ATPase activity 

kinesin-1 activation 

•Step 1: Motor binding to vesicle receptor unfolds kinesin, activating ATPase activity.

•Step 2: Transports cargo toward the (+) end of a microtubule.

kinesin-1 cargo release

not yet clear how the motor dissociated from the cargo folds back into the inhibited state

myosin and kinesin

• similar catalytic core structures

• similar myosin-II and kinesin-1 lever arms 

• but no sequence conservation 

convergent evolution has twice generated a

fold that hydrolyzes ATP to generate mechanical work

cytoplasmic dynein 

• Large protein with a globular, force-generating head.

• It is a minus end-directed microtubular motor.

• Requires an adaptor (dynactin) to interact with membrane-bounded cargo.

• Dynactin links dynein to cargo and regulates dynein activity

power stroke of dynein force-gernation mechanism

ATP-dependent change in the position of the linker causes movement of the microtubule-binding stalk 

kinesis and dyneins cooperate in the transport of

organelles throughout the cell

•(red) Cytoplasmic dyneins mediate retrograde transport of organelles toward MT(−) ends of microtubules at the centrally-located MTOC.

•(purple) Kinesins mediate anterograde transport toward MT(+) ends at the cell periphery.

•Motors reaching the end of a MT get carried back on organelles moved in the reverse direction by the other motor type.

cilia/flagella are cell-surface projections with

a central pair of singlet MTs and nine outer doublet Mts

axonemal dynein motors attached to A tubule on one doublet produce

force on the B tubule of another to bend cilic and flagella

an intraflagellar Transport (IFT) system transports

material to the tip by kinesin-2 activity and from the tip back to the base by cytoplasmic dynein activity

a nonmotile primary cilium on most cells functions as

a single atennna 

mitotic spindle

movement of chromosomes

cilia and flagella

motile appendages that project from cells

–Propel single cells

–Move fluid over cell surface in multicellular tissues

–They have similar structures but different motility.

–Cilia tend to occur in large numbers on a cell’s surface.

the structure od cilia and flagella contains a

central core (axoneme) consisting of microtubules in a ( + 2 arrangement

the basic structure of the axoneme includes

a central sheath, connected to the A tubules of peripheral doublets by radial spokes that prevent structural collapse during bending 

cilia and flagella - the doublet interconnected to one another by an inter doublet bridge composed on nexin- 

connects adjacent outer doublet microtubules to each other and prevents overslidign of adjacent doublets with respect to each other 

flagella exhibit a variety of different

beating patterns (waveforms), depending on the cell type

sperm flagella display successive

bending waves that push agains the fluid and propel the cell forward

single-celled alga pulls itself forward by

waving its two flagella in an asymmetrical manner that resembles the breaststroke of a human swimmer 

degree of asymmetry in a pattern of the beat in the algal cell is regulated by 

the internal calcium ion concentration 

movement dependent on 

Ca2+ and dynein 

cilia and flagella emerge from

basal bodies

the growth of an axoneme occurs at the

plus ends of microtubules

intraflagella transport (IFT) is the process resonsible for

assembling and maintaining flagella

IFT depends on the activity of 

both the plus and minus end-directed microtubules 

non polar fibrous filaments composed of

five classes of IF proteins

four IF classes show

tissue-specific expression and functions

class V lamina underlie and support the

membrane structure all eukaryotic nuclei

lamins interact with chromosomes inside the nucleus and

through connecting proteins with cytoskeleton in the cytoplasm

intermediate filaments (IFs) -

heterogeneous group of proteins, divided into 5 major classes

IFs classes I-IV are used in

the construction of filaments

• type V (lamins) are present in the inner lining of the nucleus

IFs radiate through the cytoplasm of a wide variety of animals cells and are often 

interconnected to other cytoksletal filaments by thin, wispy cross-bridges 

human genome has 

70 IF genes encoding proteins in at least five subfamilies 

IF assembly

Basic building block is a rod-like tetramer formed by two antiparallel dimers.

Both the tetramer and the IF lack polarity.

IF proteins structure 

•Conserved coiled-coil core domain

•Subfamily-specific globular heads and tails

•Form parallel dimers through coiled-coil core domains