[Show abstract][Hide abstract] ABSTRACT: Axonal transport mediated by microtubule-dependent motors is vital for neuronal function and viability. Selective sets of cargoes, including macromolecules and organelles, are transported long range along axons to specific destinations. Despite intensive studies focusing on the motor machinery, the regulatory mechanisms that control motor-cargo assembly are not well understood. Here we show that UNC-51/ATG1 kinase regulates the interaction between synaptic vesicles and motor complexes during transport in Drosophila. UNC-51 binds UNC-76, a kinesin heavy chain (KHC) adaptor protein. Loss of unc-51 or unc-76 leads to severe axonal transport defects in which synaptic vesicles are segregated from the motor complexes and accumulate along axons. Genetic studies show that unc-51 and unc-76 functionally interact in vivo to regulate axonal transport. UNC-51 phosphorylates UNC-76 on Ser(143), and the phosphorylated UNC-76 binds Synaptotagmin-1, a synaptic vesicle protein, suggesting that motor-cargo interactions are regulated in a phosphorylation-dependent manner. In addition, defective axonal transport in unc-76 mutants is rescued by a phospho-mimetic UNC-76, but not a phospho-defective UNC-76, demonstrating the essential role of UNC-76 Ser(143) phosphorylation in axonal transport. Thus, our data provide insight into axonal transport regulation that depends on the phosphorylation of adaptor proteins.
Full-text · Article · Jan 2009 · Genes & Development
[Show abstract][Hide abstract] ABSTRACT: Kinesin-1 is the founding member of a superfamily of motor proteins that transport macromolecules along microtubules in an
ATP-dependent manner. Classic studies show that kinesin-1 binds to intracellular cargos through non-covalent interactions
with proteins on the cargo surface, that protein–protein interaction domains are present in the cargo-binding tail domain
and that phosphorylation-dependent signal transduction pathways regulate kinesin–cargo interactions. A combination of genetics,
biochemistry and proteomics has identified processes in which kinesin-1 has an important role, and helped reveal the mechanisms
of kinesin-dependent transport events. These approaches have identified more than 35 proteins that bind to kinesin-1; these
proteins act as cargos, cargo receptors and regulators of kinesin-1 activity. This review summarizes our current understanding
of kinesin-1 associated proteins, and places those protein–protein interactions into the context of kinesin-1 in vivo function.
Preview · Article · Apr 2006 · Briefings in Functional Genomics and Proteomics
[Show abstract][Hide abstract] ABSTRACT: Motor-driven transport along microtubules is a primary mechanism for moving and positioning organelles. How such transport is regulated remains poorly understood. For lipid droplets in Drosophila embryos, three distinct phases of transport can be distinguished. To identify factors regulating this transport, we biochemically purified droplets from individual phases and used 2D gel analysis to search for proteins whose amount on droplets changes as motion changes.
By mass spectrometry, we identified one such protein as LSD2. Similar to its mammalian counterpart Perilipin, LSD2 is responsible for regulating lipid homeostasis. Using specific antibodies, we confirmed that LSD2 is present on embryonic lipid droplets. We find that lack of LSD2 causes a specific transport defect: Droplet distribution fails to undergo the dramatic changes characteristic of the wild-type. This defect is not due to a complete failure of the core transport machinery--individual droplets still move bidirectionally along microtubules with approximately normal velocities and kinetics. Rather, detailed biophysical analysis suggests that developmental control of droplet motion is lost. We show that LSD2 is multiply phosphorylated in a developmentally controlled manner. LSD2 phosphorylation depends on the transacting signal Halo, and LSD2 can physically interact with the lipid-droplet-associated coordinator Klar, identifying LSD2 as a central player in the mechanisms that control droplet motion.
LSD2 appears to represent a new class of regulators, a protein that transduces regulatory signals to a separable core motor machinery. In addition, the demonstration that LSD2 regulates both transport and lipid metabolism suggests a link between lipid-droplet motion and lipid homeostasis.
[Show abstract][Hide abstract] ABSTRACT: The microtubule-based motor kinesin-I is essential for the intracellular transport of membrane-bound organelles in the Drosophila nervous system and female germ line. A number of studies have demonstrated that kinesin-I binds to its intracellular cargos through protein-protein interactions between the kinesin tail domain and proteins on the cargo surface. To identify proteins that mediate or regulate kinesin-cargo interactions, we have performed yeast two-hybrid screens of a Drosophila embryonic cDNA library, using the tetratricopeptide repeats of the kinesin light chain and amino acids 675-975 of the kinesin heavy chain as baits. One of the proteins we have identified is YETI. Interestingly, YETI has the unique ability to bind specifically to both subunits of the kinesin tail domain. An epitope-tagged YETI fusion protein, when expressed in Drosophila S2 cultured cells, binds to kinesin-I in copurification assays, suggesting that YETI-kinesin-I interactions are context-independent. Immunostaining of cultured cells expressing YETI shows that YETI accumulates in the nucleus and cytosol. YETI is evolutionarily conserved, and its yeast homolog (AOR1) may have a role in regulating cytoskeletal dynamics or intracellular transport. Collectively, these results demonstrate that YETI interacts with both kinesin subunits of the kinesin tail domain, and is potentially involved in kinesin-dependent transport pathways.
Preview · Article · Jan 2004 · Biology of the Cell
[Show abstract][Hide abstract] ABSTRACT: Kinesin-I is essential for the transport of membrane-bound organelles in neural and nonneural cells. However, the means by which kinesin interacts with its intracellular cargoes, and the means by which kinesin-cargo interactions are regulated in response to cellular transport requirements are not fully understood. The C terminus of the Drosophila kinesin heavy chain (KHC) was used in a two-hybrid screen of a Drosophila cDNA library to identify proteins that bind specifically to the kinesin tail domain. UNC-76 is an evolutionarily conserved cytosolic protein that binds to the tail domain of KHC in two-hybrid and copurification assays, indicating that kinesin and UNC-76 form a stable complex in vivo. Loss of Drosophila Unc-76 function results in locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants, suggesting that UNC-76 is required for kinesin-dependent axonal transport. Unc-76 exhibits dosage-sensitive genetic relationships with Khc and Kinesin light chain mutations, further supporting the hypothesis that UNC-76 and kinesin-I work in a common transport pathway. Given the interaction of FEZ1, the mammalian homolog of UNC-76, with protein kinase Czeta, and the role of FEZ1 in axon outgrowth, we propose that UNC-76 helps integrate kinesin activity in response to transport requirements in axons.
Preview · Article · Sep 2003 · Molecular Biology of the Cell
[Show abstract][Hide abstract] ABSTRACT: A broadly conserved membrane-associated protein required for the functional interaction of kinesin-I with axonal cargo was identified. Mutations in sunday driver (syd) and the axonal transport motor kinesin-I cause similar phenotypes in Drosophila, including aberrant accumulations of axonal cargoes. GFP-tagged mammalian SYD localizes to tubulovesicular structures that costain for kinesin-I and a marker of the secretory pathway. Coimmunoprecipitation analysis indicates that mouse SYD forms a complex with kinesin-I in vivo. Yeast two-hybrid analysis and in vitro interaction studies reveal that SYD directly binds kinesin-I via the tetratricopeptide repeat (TPR) domain of kinesin light chain (KLC) with K(d) congruent with 200 nM. We propose that SYD mediates the axonal transport of at least one class of vesicles by interacting directly with KLC.
[Show abstract][Hide abstract] ABSTRACT: In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.
Preview · Article · Dec 1999 · Molecular Biology of the Cell
[Show abstract][Hide abstract] ABSTRACT: Kinesin is a heterotetramer composed of two 115-kD heavy chains and two 58-kD light chains. The microtubule motor activity of kinesin is performed by the heavy chains, but the functions of the light chains are poorly understood. Mutations were generated in the Drosophila gene Kinesin light chain (Klc), and the phenotypic consequences of loss of Klc function were analyzed at the behavioral and cellular levels. Loss of Klc function results in progressive lethargy, crawling defects, and paralysis followed by death at the end of the second larval instar. Klc mutant axons contain large aggregates of membranous organelles in segmental nerve axons. These aggregates, or organelle jams (Hurd, D.D., and W.M. Saxton. 1996. Genetics. 144: 1075-1085), contain synaptic vesicle precursors as well as organelles that may be transported by kinesin, kinesin-like protein 68D, and cytoplasmic dynein, thus providing evidence that the loss of Klc function blocks multiple pathways of axonal transport. The similarity of the Klc and Khc (. Cell 64:1093-1102; Hurd, D.D., and W.M. Saxton. 1996. Genetics 144: 1075-1085) mutant phenotypes indicates that KLC is essential for kinesin function, perhaps by tethering KHC to intracellular cargos or by activating the kinesin motor.
Preview · Article · Apr 1998 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: The receptor tyrosine phosphatases DPTP69D and DPTP99A are expressed on motor axons in Drosophila embryos. In mutant embryos lacking DPTP69D protein, motor neuron growth cones stop growing before reaching their muscle targets, or follow incorrect pathways that bypass these muscles. Mutant embryos lacking DPTP99A are indistinguishable from wild type. Motor axon defects in dptp69D dptp99A double mutant embryos, however, are much more severe than in embryos lacking only DPTP69D. Our results demonstrate that DPTP69D and DPTP99A are required for motor axon guidance and that they have partially redundant functions during development of the neuro-muscular system.