The Association with, and Movement of Vesicles/Organelles on Microtubules.
Microtubules are ideally suited as tracks to deliver vesicles and organelles to distant cellular regions. They are rigid and so comparatively straight, they are polar with the plus end almost invariably pointing to the periphery. Two main groups propel vesicles and organelles along MT in the cytoplasm; cytoplasmic dynein and a more diverse group, the kinesins
Cytoplasmic dynein is also known as MAP1C, it is a huge, 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. The enormous size of the dynein molecule has meant that our understanding of its structure has not progressed as fast as kinesin. The heavy chain is 4644 amino acids in rat and smaller in both Dictyostelium and Saccharomyces, about 3300 AA of this form the “head” while the remainder form the stalk (compared to 340 AAs for the kinesin head and 850 AAs for myosin). The dynein heavy chain contains 4 conserved nucleotide-binding “P-loop” motifs. Only the first of these P-loops is actually involved in nucleotide hydrolysis, but the others may also bind nucleotide, perhaps to regulate the molecule (Mocz & Gibbons, 1996). It is known that vanadate is capable of cleaving the dynein (and other ATPases) at the ATP binding site, this is, as expected in the first P-loop. The microtubule binding site is exposed where one might imagine it would be on the three dimensional structure of the dynein molecule, at the top, on an extended structure (see part a) (Gee et al, 1997). However, the microtubule site is encoded by a stretch of sequence toward the C-terminus, next to the stalk domain. Solving the 3D structure of the dynein molecule will be a major task! Not much is presently known about the regulation of dynein at the molecular level. What is known is largely phenomenological, for example it is known that okadaic acid (an inhibitor of phosphatases PP1 and PP2A) causes a 27 fold increase in the number of ER tubules moving on microtubules in an in vitro reconstituted experiment (Allan, 1995), but it is not known which of the many proteins in the complex that gets phosphorylated is the regulating component.
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. The kinesin head domain has a fold with suprising similarity to the myosin head and some switch type G-proteins (Vale, 1996). Although the kinesins carry vesicles it is suspected that each type carries a specific sub-set of vesicle. Kinesin moves vesicles at around 1 µm/sec, is inhibited by NEM and Vanadate and can only utilise ATP for force production
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 (see below).
Protein direction Mol.Wt (kDa) Speed Cargo
of heavy chain (µm/min)
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
Rabkinesin-6 ? ~100 (?) ? Golgi & TGN
ncd minus ~100 chromosomes
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.
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.
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 (see Nixon 1998 for a recent review of transport of cytoskeletal components).
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 (see Burlhardt, 1996). 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 complex (see left). At least two mRNAs and therefore two proteins are produced from the kinectin gene in humans and this may allow a measure of specificity. Early evidence suggests that kinectin is present of E.R. but not Golgi complex membranes.
The Golgi complex membrane skeleton and motor proteins.
It has very recently come to light that the Golgi complex has its own, Golgi-specific isoforms of a number of proteins involved with the actin (and microtubule to a lesser extent) cytoskeleton:-
Spectrin a linker between other membranous proteins.
Ankyrin Binds 4.1 type proteins and spectrin
ARP1 a component of the dynactin complex
Comitin an “annexin” actin and phospholipid binding protein
Other Golgi-specific protein isoforms are also expected to exist (Beck & Nelson, 1998), permitting the postulation of a scheme whereby dynein may be targeted to Golgi membranes (see left). The dynactin complex is composed of a short “filament” of 9 ARP1 molecules. ARP1 is an actin related protein that is known to bind Golgi spectrin and so it is tempting to speculate that this may form the basis for a dynein receptor. Additionally, S-100 proteins bind CapZ which it turn binds the ARP1 mini-filament, CapZ also binds phosphatidyl- inositol 4,5-bisphosphate which may also be relevant. The binding partner for golgi specific ankyrin has not yet been identified, but there are many splice variants of Band4.1 proteins known to bind ankyrin in other membranes and there are many Band4.1 related proteins.
Regulation and specificity mediated by G-proteins
Many events such as fusion, and recognition of membranes is brought about by an enormous family of G-proteins, particularly the G proteins of the rab family (see Martinez & Goud 1998). Rab6 is just one of these numerous proteins, it is restricted to Golgi and the TGN, recently, a kinesin related protein which binds to Rab6 has been identified (Rabkinesin-6; Echard et al, 1998) so it seems possible that the plus end directed kinesin-like activity already detected in the golgi, is localised and controlled by Rab6. As Rabkinesin-6 has very limited homology to the other kinesins, it is possible that there are many of these in the genome that are also too little conserved to be recognised in EST libraries and other sequencing databases.
Why are organelles arranged and moved along MTs and MFs?
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.
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 a myosin I-like activity (at least in yeast, see bottom of page 7 on previous handout). Also yeast cell with disabled kinesin are rescued by overexpression of myosin I! The sea urchin coelomocyte has been mentioned (page 6) in connection with transport of vesicles towards the cell periphery upon stimulation. In addition to using microfilaments as track, these same cells use microtubules to transport mitochondria, in fact the motility of the mitochondria is increased 1.5 fold in the absence of actin filaments suggesting that the presence of the filaments otherwise impedes there transport possibly as a result of the increased cytosolic viscosity (Krendel et al, 1998). The two motor protein tracks, microfilaments and microtubules support seemingly exactly the same type of vesicular traffic in different cell type. Melanin containing vesicles for example, are transported by Myosin V along microfilaments in mammalian melanoma cells while they are moved along microtubules in fish skin. Two very similar organisms, Reticulamyxa and Laberinthula also demonstrate this doublicity. Both organism are large syncytical “amoeba” living in fresh and salt water respectively. Reticulamyxa moves its nuclei and other organelles around on a vast arrays of microtubule bundles, while Laberinthula has bundles of actin to perform the same function.
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.
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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