Drosophila kinesin: characterization of microtubule motility and ATPase.

Saxton, William M.; Porter, Mary E.; Cohn, Stanley A.; Scholey, Jonathan M.; Raff, Elizabeth C.; McIntosh, J. Richard

doi:10.1073/pnas.85.4.1109

Cited by 105 publications

(64 citation statements)

References 22 publications

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“…Protein A-or G-coated beads (Santa Cruz Biotechnology, Santa Cruz, CA) were added, and the suspensions were rocked overnight at 4°C and then washed three times with 500 l of extraction buffer and boiled in SDS-PAGE sample buffer. SDS-PAGE (7.5% gels), blotting, and staining were done as described previously (Saxton et al, 1988;Gertler et al, 1995), except that an ECL system was used for detection (Amersham, Piscataway, NJ). Primary antibodies used to detect Ena and Khc included rabbit anti-Khc R1.5 (Saxton et al, 1988) and rabbit anti-Ena-amino-terminal (Gertler et al, 1989), as well as the antibodies described above.…”

Section: Immunoprecipitationmentioning

confidence: 99%

“…SDS-PAGE (7.5% gels), blotting, and staining were done as described previously (Saxton et al, 1988;Gertler et al, 1995), except that an ECL system was used for detection (Amersham, Piscataway, NJ). Primary antibodies used to detect Ena and Khc included rabbit anti-Khc R1.5 (Saxton et al, 1988) and rabbit anti-Ena-amino-terminal (Gertler et al, 1989), as well as the antibodies described above. Prestained molecular weight standards were included in the SDS-PAGE, and their positions were marked on blots before blocking and antibody incubations.…”

Section: Immunoprecipitationmentioning

confidence: 99%

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Abl Tyrosine Kinase and Its Substrate Ena/VASP Have Functional Interactions with Kinesin-1

Martin

Ahern-Djamali

Hoffmann

et al. 2005

MBoC

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Relatively little is known about how microtubule motors are controlled or about how the functions of different cytoskeletal systems are integrated. A yeast two-hybrid screen for proteins that bind to Drosophila Enabled (Ena), an actin polymerization factor that is negatively regulated by Abl tyrosine kinase, identified kinesin heavy chain (Khc), a member of the kinesin-1 subfamily of microtubule motors. Coimmunoprecipitation from Drosophila cytosol confirmed a physical interaction between Khc and Ena. Kinesin-1 motors can carry organelles and other macromolecular cargoes from neuronal cell bodies toward terminals in fast-axonal-transport. Ena distribution in larval axons was not affected by mutations in the Khc gene, suggesting that Ena is not itself a fast transport cargo of Drosophila kinesin-1. Genetic interaction tests showed that in a background sensitized by reduced Khc gene dosage, a reduction in Abl gene dosage caused distal paralysis and axonal swellings. A concomitant reduction in ena dosage rescued those defects. These results suggest that Ena/VASP, when not inhibited by the Abl pathway, can bind Khc and reduce its transport activity in axons. INTRODUCTIONThe activities of the F-actin and microtubule cytoskeletons are linked in cellular processes such as secretion, cytokinesis, and axon outgrowth, but relatively little is known about mechanisms that coordinate the activities of the two filament systems. The nonreceptor tyrosine kinase Abl, aberrant forms of which are implicated in human leukemia, influences axon outgrowth and other F-actin-dependent processes (Woodring et al., 2003;Hernandez et al., 2004). Recent work suggests that Abl also influences microtubule polymerization in the axon growth cone via interactions with Orbit and that a mouse Abl-related protein, Arg, can crosslink F-actin with microtubules in the cell periphery (Lee et al., 2004;Miller et al., 2004). In part, the influence of Abl on F-actin-dependent processes is mediated by its regulatory interaction with Ena/VASP proteins, which can modulate actin filament length, branching pattern, and bundle formation (reviewed by Krause et al., 2003;Kwiatkowski et al., 2003). Ena/VASP proteins have three conserved regions. The N-terminal EVH1 domain (133 amino acids, Gertler et al., 1996) can bind the focal-adhesion proteins vinculin and zyxin, as well as the growth-cone guidance receptor Robo/ Sax3. The central proline-rich region can bind profilin, which facilitates the addition of G-actin monomers to F-actin plusends. It can also bind Abl and other SH3-domain proteins. The C-terminal EVH2 domain (226 amino acids, Gertler et al., 1996) has both G-and F-actin binding sites and has been shown to mediate Ena/VASP multimerization (reviewed by Krause et al., 2003;Kwiatkowski et al., 2003).In Drosophila development, Ena and Abl are known to have an antagonistic relationship. Zygotic mutations in the ena or Abl gene cause axon growth-cone guidance defects and lethality, but normal axon guidance and viability can be restored by combining ena and Ab...

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Section: Immunoprecipitationmentioning

confidence: 99%

Section: Immunoprecipitationmentioning

confidence: 99%

Abl Tyrosine Kinase and Its Substrate Ena/VASP Have Functional Interactions with Kinesin-1

Martin

Ahern-Djamali

Hoffmann

et al. 2005

MBoC

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“…All Drosophila cells tested to date express KHC (Saxton et al, 1988). Tests of eye and wing discs by immunofluorescence (Golic and Lindquist, 1989;Golic, 1991;Xu and Rubin, 1993).…”

Section: Kinesin Is Dispensable For the Growth And Division Of Undiffmentioning

confidence: 99%

“…Conventional kinesin (often referred to below simply as kinesin) is a ubiquitous and abundant plus end-directed (Saxton et al, 1988;Hollenbeck, 1989). The majority of kinesin appears to be free in cytosol, but various studies have shown that it can associate with ER, vesicles, mitochondria, and other organelles (Hollenbeck, 1989;Pfister et al, 1989;Brady et al, 1990;Hirokawa et al, 1991;Houliston and Elinson, 1991;Wright et al, 1991;Leopold et al, 1992;Yu et al, 1992;Marks et al, 1994;Elluru et al, 1995; Okada et al, 1995;Sturmer and Baumann, 1996).…”

Section: Introductionmentioning

confidence: 99%

“…In some but not all polarized epithelial cells, microtubules are oriented with their plus ends toward the basal pole and their minus ends near the apical pole (reviewed by Mays et al, 1994;McNiven and Marlowe, 1999). In such situations, in which the polarity is relatively uniform, models have been developed that invoke appropriate microtubule motors for various steps in vesicle or other organelle transport.Conventional kinesin (often referred to below simply as kinesin) is a ubiquitous and abundant plus end-directed (Saxton et al, 1988;Hollenbeck, 1989). The majority of kinesin appears to be free in cytosol, but various studies have shown that it can associate with ER, vesicles, mitochondria, and other organelles (Hollenbeck, 1989;Pfister et al, 1989;Brady et al, 1990;Hirokawa et al, 1991;Houliston and Elinson, 1991;Wright et al, 1991;Leopold et al, 1992;Yu et al, 1992;Marks et al, 1994;Elluru et al, 1995; Okada et al, 1995;Sturmer and Baumann, 1996).…”

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confidence: 99%

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Clonal Tests of Conventional Kinesin Function during Cell Proliferation and Differentiation

Brendza

Sheehan

Turner

et al. 2000

MBoC

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Null mutations in the Drosophila Kinesin heavy chain gene (Khc), which are lethal during the second larval instar, have shown that conventional kinesin is critical for fast axonal transport in neurons, but its functions elsewhere are uncertain. To test other tissues, single imaginal cells in young larvae were rendered null for Khc by mitotic recombination. Surprisingly, the null cells produced large clones of adult tissue. The rates of cell proliferation were not reduced, indicating that conventional kinesin is not essential for cell growth or division. This suggests that in undifferentiated cells vesicle transport from the Golgi to either the endoplasmic reticulum or the plasma membrane can proceed at normal rates without conventional kinesin. In adult eye clones produced by null founder cells, there were some defects in differentiation that caused mild ultrastructural changes, but they were not consistent with serious problems in the positioning or transport of endoplasmic reticulum, mitochondria, or vesicles. In contrast, defective cuticle deposition by highly elongated Khc null bristle shafts suggests that conventional kinesin is critical for proper secretory vesicle transport in some cell types, particularly ones that must build and maintain long cytoplasmic extensions. The ubiquity and evolutionary conservation of kinesin heavy chain argue for functions in all cells. We suggest interphase organelle movements away from the cell center are driven by multilayered transport mechanisms; that is, individual organelles can use kinesin-related proteins and myosins, as well as conventional kinesin, to move toward the cell periphery. In this case, other motors can compensate for the loss of conventional kinesin except in cells that have extremely long transport tracks. INTRODUCTIONVesicle transport is important in eukaryotic cells for the addition of material to the plasma membrane, for secretion, and for cell polarity. Active vesicle transport is thought to be driven by mechanochemical enzymes (motor proteins). Motors attach to vesicle membranes and then use ATP hydrolysis to drive unidirectional movement along polar cytoskeletal filaments. Characterized members of the myosin family of motors move toward the barbed or fast-growing ends of actin filaments with the exception of myosin VI, which moves toward the pointed or slow-growing ends (Wells et al., 1999) (reviewed by Sellers and Goodson, 1995). Actin filaments in undifferentiated and in some differentiated cell types are highly concentrated in the cortex (WatermanStorer et al., 1998; reviewed by Cramer, 1999). Cytoplasmic myosins may therefore be important for anchoring or moving vesicles when they are near the plasma membrane (Fath et al., 1994). Motors in the kinesin and dynein families move along microtubules. Characterized dyneins and members of the C-terminal kinesin subfamily move toward microtubule minus ends, whereas other kinesins that act as motors move toward microtubule plus ends (reviewed by Vale and Fletterick, 1997;Hirokawa, 1998;Goldstein and Ya...

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Kinesin proteins: A phylum of motors for microtubule‐based motility

1996

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The cellular processes of transport, division and, possibly, early development all involve microtubule-based motors. Recent work shows that, unexpectedly, many of these cellular functions are carried out by different types of kinesin and kinesin-related motor proteins. The kinesin proteins are a large and rapidly growing family of microtubule-motor proteins that share a 340-amino-acid motor domain. Phylogenetic analysis of the conserved motor domains groups the kinesin proteins into a number of subfamilies, the members of which exhibit a common molecular organization and related functions. The kinesin proteins that belong to different subfamilies differ in their rates and polarity of movement along microtubules, and probably in the particles/organelles that they transport. The kinesins arose early in eukaryotic evolution and gene duplication has allowed functional specialization to occur, resulting in a surprisingly large number of different classes of these proteins adapted for intracellular transport of vesicles and organelles, and for assembly and force generation in the meiotic and mitotic spindles.

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Drosophila kinesin: characterization of microtubule motility and ATPase.

Cited by 105 publications

References 22 publications

Abl Tyrosine Kinase and Its Substrate Ena/VASP Have Functional Interactions with Kinesin-1

Abl Tyrosine Kinase and Its Substrate Ena/VASP Have Functional Interactions with Kinesin-1

Clonal Tests of Conventional Kinesin Function during Cell Proliferation and Differentiation

Kinesin proteins: A phylum of motors for microtubule‐based motility

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