Cell Motility and the Cytoskeleton- Microtubules CS4

Microtubules are ideally suited as tracks to deliver vesicles and organelles to distal cellular regions. They are rigid and so comparatively straight, they are polar with the plus end almost invariably pointing to the periphery. Intracellular transport and motility is mediated by MTs, but there is no direct involvement in crawling cell locomotion. Although cells with a highly developed microtubule network such as fibroblasts and newt eosinophils become depolarised and loose speed and directionality upon disruption of microtubules, it is likely that microtubules disruption causes concomitant changes in the microfilament system. Cells that are naturally devoid of cytoplasmic microtubules (e.g. Naegleria) are capable of efficient and rapid locomotion. MTs are abundant and crucial components of the flagella which drives the swimming locomotion of eukaryotic cells. A multitude of MT-based motors drives the chromosome reorganisation at mitosis and meiosis. Two main groups propel vesicles and organelles along MT in the cytoplasm; cytoplasmic dynein and a more diverse group, the kinesins

Cytoplasmic Dynein

Cytoplasmic dynein is also known as MAP1C, it is a large two headed ATP-ase which is minus end directed. Some dyneins from the flagella of Chlamydomonas and Tetrahymena have three heads, but most other flagellar dyneins have just the two. Cytoplasmic dynein moves vesicles at around 0.3 µm/sec in vitro, is unaffected by NEM and Vanadate and can utilise ATP, GTP or ITP for force production.

Figure 18

The Kinesins

The kinesins are a large group of microtubule based motor proteins which are formed from multi-component complexes. The first kinesin was identified in the axon in 1985 but several other kinesin related proteins (KRP) have come to light. Three kinesin subfamilies have been implicated in the plus end directed movement of vesicles and organelles. All three share a similar kinesin “head” domain but are otherwise quite different. Although they all seem to carry vesicles it is suspected that each carries a specific type of vesicle. Kinesin moves vesicles at around 1.25 µm/sec, is inhibited by NEM and Vanadate and can only utilise ATP for force production

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Protein direction Mol.Wt (kDa) Speed Cargo

of heavy chain (µm/min)

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Kinesin plus 110 (heterotetrameric) 30-54 Vesicles

Unc-104 (KIF1A) plus 192 (monomeric) 72 precursor vesicles

(KIF1B) plus 130 (monomeric) 40 Mitochondria

Kinesin II (KRP85/95) plus 79 (heterotrimer) 24 Vesicles

Many other kinesin related motor proteins have been discovered which are involved in the spindle pole formation and karyokinesis. These have largely been identified at the gene level by genetic screen and so not much is known about their biochemical properties. However, one of these, NCD is a kinesin related protein which drives minus end movement!

Figure 19

Control of Dynein and Kinesin motor activity.

In most cell types, MTs have their plus ends facing the plasma membrane and the minus end associated with a MTOC deep in side the cell. Kinesins will therefore generally move organelles toward the plasma-membrane and dyneins take them towards the nucleus. A spectacular example of the is the movement of pigment granules along the MT in the chromatophore from the skin of a variety of fish and amphibians. Chromophores are stellate flattened cell types which contain a multitude of dense light absorbing granules. When the granules are packed in the centre of the cell, the skin appears light, but darkens when the pigment granules are dispersed throughout the cell. This affords the animal protective camouflage. Granule movement requires microtubules. Dispersion depends on cAMP or IP3/diacylglycerol, the activity of PKA or PKC, and kinesin. Aggregation depends on phosphatase activity and it thought to be dynein dependent, although this step is very rapid at 5µm/sec and dynein driven motility is only 0.3 µm/sec in vitro. Cellular cofactors absent from the in vitro studies may account for this difference.

Figure 20

Figure 21

Other membranous organelles such as the Gogli, RER, lysosomes and mitochondria are moved around the cell on microtubules. Many of these share the same motors so it is difficult to see how the cell switches on motility of one particular organelle and not others. Perhaps motor receptors are the key (see later), localised phosphorylation of motor protein complexes is perhaps another controlling mechanism.

Figure 22

Transport of material along the nerve axon. Materials such as neurotransmitter peptides are synthesised in the cell body and sequestered in vesicles at the golgi. These vesicles are then transported down the axon towards the synapse by kinesin motor proteins. This distance may be yards in the case of a giraffe sciatic nerve! Other materials are transported from the synapse to the cell body by dynein motors.

Co-operation between Microtubules and Microfilament systems in vesicular transport

In many cases there appears to be considerable overlap in the vesicular motility driven by the MT and MF systems. Mitochondria are moved along MTs by KIF1B, and along MFs at 1.4µm/min by an unidentified myosin I-like activity (at least in yeast). Also yeast cell with disabled kinesin are rescued by overexpression of myosin I!

Are there specific Kinesin and Dynein receptors on cargo membranes?

Recently a putative receptor “kinectin” has been identified by passing detergent-solubilized microsomal membranes over a kinesin affinity column. Kinectin has a molecular weight of 160 kDa and the cloned cDNA reveals that the protein has a hydrophobic N-terminus and a high probability of forming coiled-coil Kinectin has numerous putative phosphorylation sites and is known to be phosphorylated on serine, however there has been no correlation between the state of phosphorylation and kinesin binding activity to date. Kinectin is most abundant on the ER membranes, and is not thought to bind dynein. It is very likely that receptors other than kinectin exist in or on other membranes. A possible candidate for a dynein receptor is “dynactin”, a 150-170 kDa protein.

Why are organelles arranged and moved along MTs and MFs?

When a cell divides in two each half requires the correct amount of organelle activity to be included in its share. Organelles such as mitochondria are only made from existing mitochondria and cannot be synthesised de novo. If a daughter cell finds itself without enough mitochondria, it may not be capable of producing all its energy requirements and so may die before being able to grow more mitochondria. The microfilament and microtubule system is shared very equitable between daughter cells because they constitute the very apparatus which separates them, thus if organelles are arranged on these structures this will guarantee an equitable disruption of organelles between daughter cells. Other cell type have got around this problem using different means. A large amoeba Pelomyxa palustris does not have mitochondria, but does have symbiotic bacteria which attach themselves to the nuclear membrane during cytokinesis. (This works because unlike higher eukaryotes the nuclear membrane does not break down).

Organelles are required to be unequally distributed in certain situations and so motor proteins and tracks are needed to set up and maintain this distribution against chaotic influences. Secretary vesicles, for example are required only at the synapse and so are taken there by axonal transport (by kinesin). The golgi apparatus performs an assembly line function where proteins are processed in a linear fashion one modification taking place only after another is completed. This process is made more efficient by having specific vesicles arranged on microtubules in the cis - trans configuration with intermediate vesicles shuttling products between them.

Why have no Motor Proteins associated with Intermediate Filaments been discovered?

In order for a motor protein to do useful work, some directing influence must exist. In the case of the other two systems MT and MF, the motors are directed my the polarity of the polymers, however IFs are not polar and so it is difficult (but not impossible) to imagine how a motor could proceed along an IF in one direction Motor proteins evolved very early in the Eukaryotes and today many protozoans express a great variety of MT and MF motor proteins, however, the IF system is much more recent (probably) being absent from the protozoan and possibly from the arthropods. Consequently, it may be that IFs have not been around long enough for specific IF motor proteins to evolve, also this function would seem to be adequately fulfilled by MTs and MFs.

References:-

Microtubule Motors Barton, N.R. & Goldstein, L.S.B. “Going mobile: Microtubule motors and chromosome

segregation.” Proc.Nat.Acad.Sci. USA. March 1996. 93, 1735-1742.

Goodson HV, Valetti, C & Kreis, T.E. Current Opinion in Cell Biol. Feb. 1997. 9, 18-28.

MT & MF motors Allan, V. “Membrane traffic motors” FEBS letters 369, 101 1995.

Kinesin Scholey, J.M. “Kinesin-II, a membrane traffic motor in axons, axonemes, and spindles.”

Journal of Cell Biology April 1996. 133, 1-4.

Moore, J.D. & Endow, S.A “Kinesin proteins: a phylum of motors for microtubule-

based motility” BioEssays 18 207, April 1996.

Brady, S.T. “A kinesin medley: biochemical and functional heterogeneity” Trends in Cell Biology

5, 159 April 1995.

Please direct any questions to me at:-

Room 444 or lab 446 fourth floor HRB. Tel (0131) 650 3714 or 3712. E-mail SKM@srv4.med.ed.ac.uk