Molecular Motors

Filament…….motor

Actin…….….myosins

Tubulin….….dynein and kinesins

Myosin Family Tree Is Large and Diverse

Differences in the myosin tails influence movement along the actin cytoskeleton

Kinesin and Dynein Comprise 2 classes of MT motors

Kinesins are involved in organelle transport, in mitosis, in meiosis, and in the transport of synaptic vesicles along axons. Cytoplasmic dyneins are involved in organelle transport and mitosis.

Organelles and vesicles containing kinesin move from the minus end of a microtubule (at a microtubule organizing center, such as centrosome) to the plus end (from the center of a cell to its periphery, called anterograde transport).

Cytoplasmic dynein moves the particles from the plus end to the minus end of the microtubules (retrograde transport).

Ciliary dynein is a much larger protein than the cytoplasmic dynein: its mass is about 2 million daltons, composed of either 2 or 3 heavy chains and 10 or more smaller polypeptides. The dynein motor protein in cilia and flagella has a head which hydrolyzes ATP and interacts with the adjacent microtubules to generate a sliding force between the microtubules. However, the microtubules are linked together, therefore, they can not slide but must bend. This local bending of the microtubules is the mechanism of ciliary movement, the beating.

The structural similarity between myosin and kinesin head groups indicates a common evolutionary origin

Microtubules are polymers of and tubulin

GTP-binding heterodimers that form a ring of 13 monomers

Microtubules grow faster at the plus end than the minus end

Cell was injected with rhodamine-tubulin such that 1 out of 20 tubulins was fluorescent.

Using a “dark mark” in the center of the microtubule asA reference, it’s apparent that the plus end grows andThe minus end shrinks. (Rather than the microtubuleMoving from left to right).

The plus end also shows dynamic Instability.

Dynamic Instability allows Transient MT Growth

Catastrophe is loss of GTP end(Occurs when GTP hydrolysis exceeds binding)Rescue is binding of tubulin GTP onto end

Microtubule Associated Proteins(MAPs)

-tubulin ring complex ( -TuRC) nucleates mictrtubule assembly

It’s at the MINUS end.

The effects of proteins that bind to microtubule ends

MAP kinase = mitogen activated proteinMAP = microtubule associated proteins

Katanin (Japanese for sword) is a microtubule Severing protein

Taxol-stabilized rhodamine-labelled microtubules were fixed to glass slides.

Purified katanin and ATP were added; some breaks seen after 30 sec; many breaks 3 min.

Drugs can influence cytoskeleton integrity (can stabilize or destabilize)

Chemical Synthesis of Taxol is a recent advance; used to treat cancer

Microtubule Functions

Centrioles (Basal Bodies) Promote Mitosis

Microtubules can form Cilia (Flagella) and basal bodies (centrioles)

The center of the center of the cell…Centrioles in a centrosome connect to centromeres

MTOC=microtubule organizing center

Centromeres bound to microtubules bound to centrosomes

-Small square wells were micromachined into a plastic substrate-A single centrosome was placed inside along with depolymerizedMicrotubules-As microtubules polymerize they push against the walls of the square-This leads to a centering effect on the centrosome

Self-organization as a Guiding Principle in Polarity

A GTPase can act as a MT directionality signal

The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-beta family. The Ran-importin-beta system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1. However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran–importin-beta system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-beta by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran–importin-beta–cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports, we observe that the Ran system also affects spindle pole formation and chromosome congression in vivo.

A GTPase can act as a MT directionality signal