Kinesin-1 (formerly kinesin heavy chain, KHC or conventional kinesin) is abundant in virtually all cell types, at all stages of development, and in all multicellular organisms tested. While a majority of Kinesin-1 appears to be free in the cytoplasm, some is associated with various membrane-bounded organelles, including small vesicles and endoplasmic reticulum, and membranes that lie between the ER and Golgi. Anti-Kinesin-1 antibodies known to inhibit the motor’s mechanochemical function in vitro have been injected or perfused into isolated cells. These treatments have been shown to inhibit the movement of tubular lysosomes, Golgi-derived transport vesicles, membrane-bounded pigment granules, and intermediate filament networks. In another approach, anti-sense oligonucleotides complementary to Kinesin-1 mRNA inhibit the anterograde axonal transport of various proteins. These and other data have led to acceptance of the idea that Kinesin-1 transports membrane-bounded organelles and perhaps macromolecular protein assemblies toward microtubule plus ends.
Analysis of the effects of recessive lethal mutations in the Drosophila Kinesin-1 or kinesin heavy chain (Khc) gene by my lab and our collaborators indicates that Kinesin-1 is a motor for fast axonal transport. Before dying, Khc mutant larvae suffer from progressive distal paralysis. The paralysis appears earliest and is most severe on the ventral side of the posterior segments. This causes an imbalance in body wall contractions such that larvae rhythmically flip their tails upward as they crawl (see Movie Page). This distinctive phenotype is the manifestation of a series of neuronal defects that begins with a disruption of fast axonal transport. The transport defect can be recognized in the segmental nerves of early second instar larvae by the appearance of large axonal swellings packed with membrane-bounded organelles. The random distribution and composition of these ‘organelle jams’ suggests that they are initiated by the accumulation of stalled Kinesin-1 cargoes.
As they increase in size, the organelle jams appear to act as genetically induced nerve ligations, hindering both anterograde and retrograde fast transport. This early defect in Khc mutants impairs action potential propagation in third instar larvae by reducing the activity of axonal ion channels. We presume that the organelle jams inhibit the anterograde movement of vesicles that deliver ion channels to the axonal membrane. Immunocytology suggests that organelle jams also inhibit the delivery of vesicles bearing membrane proteins, such as Fasciclin II, synaptotagmin, and syntaxin that are needed in synaptic terminals. As a result, motor axon terminals become dystrophic as larvae develop. Thus, the number of synaptic boutons per terminal in third instar larvae is reduced by a factor of five in posterior segments and by a factor of three in anterior segments. This posterior/anterior differential is paralleled by differential reductions in neurotransmitter secretion capacity, as measured by direct stimulation of excitatory junctional currents. Since the central nervous system is located in the anterior end of a larva, the axons that innervate posterior segments are much longer than those that innervate the anterior segments. Thus, it appears that impaired Kinesin-1 function inhibits axonal transport, which causes length dependent defects in axon structure and function, which in turn cause length dependent transmitter secretion defects. Finally, the secretion defects cause the progressive distal paralysis and tail flipping behavior.
The neuronal phenotypes of Khc mutants are remarkably similar to symptoms of some motor neuron diseases in humans and other vertebrates. The parallels include axonal swellings, defective compound action potentials, reduced transmitter secretion by motor terminals, axonal dystrophy, and progressive distal paralysis. Since the Drosophila phenotypes appear to be due to a discrete disruption of fast rather than slow axonal transport, the parallels suggest that defects in fast axonal transport may be a major factor in some human motor neuron diseases. Mutations in microtubule motor proteins could be responsible for some heritable forms of those diseases.
Contributed by Bill Saxton
- Hirokawa, N. (1996) Trends Cell Biol. 6, 135-141
- Gho, M., McDonald, K., Ganetzky, B. & Saxton, W.M. (1992) Science 258, 313-316
- Saxton, W.M., Hicks, J., Goldstein, L.S.B. & Raff, E.C. (1991) Cell 64, 1093-1102
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Kinesin-related proteins – Kinesin-2
Kinesin-2 (formerly kinesin-II or KRP85/95) is the name given to a heterotrimeric kinesin-related motor protein that was discovered using anti-kinesin peptide antibodies and purified from sea urchin egg cytosol by microtubule affinity, gel filtration chromatography, monoQ ion exchange chromatography and sucrose density gradient centrifugation. In a motility assay, purified Kinesin-2 moves towards the plus ends of microtubules at ~0.4 µm/sec, it has a native molecular weight of 300 kDa, and it consists of 1 molecule each of two motor subunits, KRP85 and KRP95 that are members of the Kinesin-2 (formerly kinesin-II or KRP85/95) subfamily, plus 1 molecule of KAP115, a novel kinesin accessory polypeptide (KAP). The sequences of the two motor subunits predict a domain organization similar to Kinesin-1: both polypeptides are predicted to have an amino-terminal motor domain bearing 45% sequence identity to Kinesin-1, the motor domains lie next to central stalk regions that appear to form heterodimeric coiled coils, and finally there is a second globular tail domain at the C terminus.
The sequence of the accessory subunit, KAP115, predicts a 95 kDa, 828-residue globular alpha-helical protein that bears no sequence similarity to Kinesin-1 light chains and appears to associate with the C-terminal tails of the motor subunits. This latter hypothesis is consistent with rotary-shadow electron microscopy of purified Kinesin-2 holoenzymes which comprise two 10 nm motor domains linked to a rod, 26 nm in length, terminating in a large asymmetric structure formed by the C-terminal ends of the motor subunits and KAP115. The motor domains can presumably move along a microtubule in a hand-over-hand fashion. The rod functions as a dimerization domain that drives heterodimerization of the two motor subunits of Kinesin-2, but why two different motor subunits are needed isn’t clear. The C-terminal tail of Kinesin-2 is juxtaposed to the accessory subunit, KAP115, forming a structure hypothesized to be involved in cargo binding, allowing Kinesin-2 to transport its cargo towards the plus ends of microtubules at ~0.5 µm/sec.
Sequence analysis reveals that close relatives of the motor subunits of Kinesin-2 exist in other organisms: mouse KIF3A/KIF3B and Drosophila melanogaster KLP64D/KLP68D were discovered in PCR screens, while Chlamydomonas reinhardii FLA10 and Caenorhabditis elegans Osm-3 were discovered through analysis of mutants. These motor subunits form a subfamily of motor polypeptides referred to as the Kinesin-2 (formerly kinesin-II or KRP85/95) subfamily. The mouse and Drosophila proteins have been expressed as recombinant proteins and shown to move with a similar rate and directionality as the purified sea urchin Kinesin-2 holoenzyme. It is hypothesized that all members of the Kinesin-2 family are subunits of heterotrimeric complexes, and evidence is emerging that this is indeed the case.
Kinesin-2 is thought to drive membrane-associated movements in axons, axonemes, and melanophores. For example, C. elegans Osm3 is required for the assembly of functional ciliated chemosensory neurons, and defects in Osm3-driven intracellular transport are associated with defects in chemosensation. C. rheinhardtii Fla10 is thought to be responsible for the intraflagellar transport of electron-dense rafts between the axoneme and the flagellar membrane, as well as the delivery of inner arm dynein to the distal ends of the axoneme. Defects in Fla10 activity cause defects in flagellar assembly and stability. Kinesin-2 was recently been identified as a motor involved in dispersion of melanosomes in fish melanophores.
Contributed by Jonathan M. Scholey
- Scholey, J.M. (1996) Kinesin-II, a membrane traffic motor in axons, axonemes and spindles. J. Cell Biol., 133: 1-4.
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Copyright 1996-2007. All rights reserved
Created 22 July 1996 16:00 GMT
Modified 13 May 2007 21:15 GMT