1 Cellular motility requires microtubules or microfilaments (or both) • Microfilaments: actin polymers • Microtubules: tubes of tubulin dimers • --Move vesicles to periphery (via kinesin) • --Move vesicles to center (via dynein)

• --Retro/Antergrade transport to/from golgi and ER • --Exocytosis/Endocytosis

• Axonemes • --Movement of cilia • --Movement of flagella

2 Fast Axonal Transport:(2 um/sec=7mm/hour): Moves vesicles to axon ending (pre-synaptic membrane)

Anterograde transport away from nucleus to (+) end Kinesin protein mediates this transport direction Kinesin requires ATP hydrolysis Synapse has no ribosomes/mRNA, poor ability to synthesize things it needs like neurotransmitters and membrane proteins that localize there.

Speed of transport FAST relative to diffusion rate

Rate of synaptic damage repair process? Why might it take 6 days to fix paralysis? 1hour/7.2 mm X 1000 mm distance = 5.6 days minimum!

GEF-H1 CELLULAR MOTORS: PRINCIPLES Rho-specific GEF inactive when associated with microtubules 1 Movement is related to the shape & polarity of the cell 2 “Track” has to: (i) be aligned (ii) be spaced (iii) be anchored (iv) have an orientation established during assembly

activated 3 Vesicles need attachment to the motor protein when microtubules are depolymerised 4 Energy has to be provided immediately 5 Signals needed for timing of the movement 6 Specific disrupters of the track can reveal the mechanism of a particular observed movement, e.g. , cytochalasin, phalloidin (actin), colchicine Krendel et al., Nat CB 4: 294 (2002) (microtubules)

3 CELLULAR MOTORS: TYPES 1-4

Kinesin Vesicle transport Microtubule associated Proteins 1 - + (MAP) MICROTUBULE

Motor Proteins Cytoplasmic dynein J Vesicle transport kinesins 2 Myosin-I dyneins ACTIN FILAMENT dynamines - + Other MAPs 3 + Axonemal dynein Microtubule sliding for ciliary & flagellar beat

ACTIN 4 - +

Filament sliding for muscle contraction heads MYOSIN-II

Vesicle Transport in Two Directions

Microtubule-associated motor proteins 1. Dynein, minus end motor 2. Kinesin, plus end motor Involved in vesicle and organelle transport, mitosis, meiosis.

4 Class Cargo Direction of Movement Cytosolic kinesins cytosolic vesicles + Spindle kinesins spindle and astral MTs, + or - centrosomes, kinetochores Cytosolic dyneins cytosolic vesicles, - kinetochores Axonemal dyneins doublet microtubules in - celia and flagella

5 6 7 8 DYNEIN

Retrograde transport carries proteins and vesicles (eg. from the synapse) back to the nucleus via dynein proteins!

• One-Way Travel toward (-) charged MT end! • Lets nucleus modify its mRNA synthesis based on activity so it knows what’s going on biochemically • Cytoplasmic Dynein: requires some special elements to grab desired vesicles! • Dynactin: grabs Dynein, Ankyrin and Spectrin • allows vesicle to adhere to dynein/MT • If you had a bacteria or polio virus hijack dynein in your nose, how long would it take to enter your brain via retrograde fast axonal transport? 100 mm X 1hour/7.2 mm = 13.9 hours

Dynein binds to some membrane proteins directly via its light chains. Binding of dynein to membranes or other cargo often involves dynactin, a large complex of dynein and associated proteins that includes the following:  dynein, including two heavy chains with motor domains, plus 3-4 each intermediate & light chains.  glued, a 150 kDa protein that has a microtubule- binding domain.  Arp1, an actin-related protein that forms short filaments of constant length (8-10 subunits), capped by other small proteins.  dynamitin, a protein may link dynein & glued to Arp1.

9 What do dynein (to – end) and kinesin (to +end) look like?

wLIS1 LIS1 protein is associated with dynein/dynactin in the cell cortex. Genetic defects in LIS1 lead to the disease lissencephaly, in which brain development is severely impaired. In animal cells, overexpression or elimination of LIS1 causes mitotic spindle abnormalities including altered spindle orientation in polarized epithelial cells.

Dynein and kinesin proteins can be active at the same time and moving vesicles in opposite directions (- or +) on the same microtubules!

10 11 Microtubule Associated Proteins Other MAPs (MAPs) MAPs speed up nucleation and stabilize microtubules.

MORE COMPLEX STRUCTURES centrioles MTOC flagellae

12 • The cell cycle and mitosis: mechanism (example) CENTRIOLES - MTOC - KINETOCHORES

nonkinetochore kinetochore microtubule microtubules

Microtubules radiate from the (-)charge origin at the MTOC “MicroTubuleOriginatingCenter” out to the (+) charge end at the periphery.

• MTOC polarizes a cells MT • Centromeres, Ciliary basal body, Axon base

• MT polarity determined by the dimers stacking • (-) origin/(+) periphery • Tubulin dimers mostly added to (+) end • Tubulin mRNA synthesis Tightly Regulated: • Tubulin represses its own mRNA synthesis

• MAPs (kinesin and dynein) allow vesicles to creep along the MT and carry vesicles, organelles, proteins to/from destinations.

13 MTOC = Microtubule Organizing Center

Microtubules: centrosome and centrioles

14 15 16 17 The axoneme has a 9 double MT + 2 center MT pattern The axonema and the dynein in it is the central with dynein arms on the outside for sliding action! structure that lets cilia and flagella bend! This is the “Sliding Filament Theory” • Basic Unit: “Axoneme”: 9+2 VIP number! 9 doublets (pairs) surrounding two center MTs Add radial spokes, interchain links and dynein! Origin: basal body or kinetosome(centriole-like) Motion: dynein slides MTs across each other

Cilia: Many short axonemes per cell Contact in coordinated fashion! Where are cilia? Airway cleaning (smoking= damage= cough?) Fallopian Tubes (damage = infertility?) Protozoans called “ciliates” Flagella are basically cells with just one long axoneme!

18 Dynein arms move across adjacent MTs and cause sliding and axonemal bendingmotion

19 BUILD A CENTRIOLE or BASAL BODY

Assemble 2 partial & one complete microtubules into a TRIPLET C 10 B subfibers 10 13 A protofilaments

Arrange 9 triplets in parallel & position an identical array nearby & perpendicular

CENTROSOME = 2 Centrioles + Microtubule Organizing Center

MICROTUBULES BUILD AN AXONEME of cilium or flagellum

Extend A & B subfibers to be the axoneme’s doublets

B 10 subfibers 13 protofilaments A

9 doublets

central pair

Other microtubule arrays are the MITOTIC SPINDLE & AXONAL CORE

20 MICROTUBULES FOR THE CILIARY BEAT

Dynein arm with ATPase activity to power movement - generating a sliding interaction with B subfiber of adjacent microtubule doublet

Connections turn the microtubule sliding into BENDING of the Cilium

MICROTUBULES FOR THE CILIARY BEAT CILIATED AIRWAY CELL

Dynein arm with ATPase 9 + 2 Microtubular array (9 doublets) activity to power movement - CILIUM generating a sliding interaction Basal bodies with 9 + 0 array (9 triplets) with B subfiber of adjacent . . Zonula occludens microtubule doublet . Zonula adherens

Macula adherens/Desmosome Connections turn the microtubule GOLGI Keratin Intermediate Filaments sliding into BENDING of the Cilium

Centrioles & MTOC MEDICAL CORRELATIONS for Microtubules A genetic axonemal dynein deficiency impairs ciliary clearance of luminal the airway, leading to severe lung infections of Kartegener’s Actin cortex syndrome lateral Hemi-desmosome

Dangerous cell proliferation in cancer can be halted by vinblastine . which blocks mitotic spindle formation BL basal

21 SENSORY STEREOCILIA ON AUDITORY HAIR CELL SENSORY STEREOCILIA ON AUDITORY HAIR CELL

bundled, as the as the core of the stereocilium Stereocilia are core of the stereocilium non-motile, Actin filaments Ion channels Actin filaments opened by but can be deflection of meshwork, as anchoring moved by the meshwork stereocilium cuticular plate stimulus Stereocilia are non-motile, Hair cell also but can be has a solitary moved by the tall true stimulus cilium, with microtubules

Synapses

SENSORY STEREOCILIA ON AUDITORY HAIR CELL Dynein arms move across bundled, as the as the core of the stereocilium adjacent MTs and cause sliding and axonemal Actin filaments bendingmotion meshwork Note: no Ion channels microtubules in the signal-transduction mechanism

& Stereocilia are non-motile

So why the ‘cilium’ name? Why, indeed!

Synapses

22 CELL SHAPES VARIETIES OF MOVEMENT

1 Cylindrical/columnar, cuboidal, polyhedral, flattened Ciliary & Flagellar Whole cell epithelial cell shapes to fit into multicellular patterns Intracellular vesicles Extension of processes 2 Spheroid & Ovoid - defensive blood cells Chromatids during mitosis Separation of cells at mitosis 3 Elongated - muscle cells & fibroblasts Whole cell (sperm) Intracellular vesicles 4 Multiple branching processes - neurons, glial cells, pigment cells by MICROTUBULES by ACTIN FILAMENTS Generalizations Generalization Little direct motor role for Intermediate Filaments Microtubules and intermediate filaments with cell junctions Microtubules & IFs highly concentrated around the nucleus & hold shape and polarization extend radially; actin more peripheral under the plasmalemma in the “cortex” Exceptions are: muscle, neurons, mature epidermal cells, RBCs Actin microfilaments (with & without myosin) modify shape Movements are closely related to cell’s shape & polarity

23 24 MICROTUBULE CONSTRUCTION a. Dimerization of a and b tubulin subtypes

b. Linear repitition of the heterodimers makes a

a b - a b - a b - a b - a b - a b - a b - a b - a b - a b + Microtubule organizing tubulin protofilament growing center c. Side-by-side assembly of 13 protofilaments to make a sheet d. Rolling of the sheet into a tubule

+ e. Elongation controlled by [ions] [a b ] GTP GDP rate

Colchicine blocks elongation

• The cell cycle and mitosis: mechanism (example) CENTRIOLES - MTOC - KINETOCHORES

nonkinetochore kinetochore microtubule microtubules

25 The axoneme has a 9 double MT + 2 center MT pattern with dynein arms on the outside for sliding action! This is the “Sliding Filament Theory”

26