Immunochemical analysis of kinesin light chain function.

Stenoien, David L.; Brady, Scott T.

doi:10.1091/mbc.8.4.675

Cited by 132 publications

(165 citation statements)

References 60 publications

(91 reference statements)

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“…Second, that plus end motility is increased when minus-end motility is inhibited by incubation of vesicles with antibody to Kifc1 (Figure 8). These data are consistent with the possibility that these motors may be part of a yet to be elucidated protein complex that mediates their coordinate regulation, as has been suggested for vesicle-associated dynein and plus-end kinesins (Brady et al, 1990;Stenoien and Brady, 1997;Waterman-Storer et al, 1997;Ligon et al, 2004). This view is supported in the present study by the finding that Kifc1 and Kif5B can interact with each other as shown by immunoabsorption of native Kif5B by FLAGKifc1 expressed in 293T cells ( Figure 9B).…”

Section: Discussionsupporting

confidence: 93%

Kif5B and Kifc1 Interact and Are Required for Motility and Fission of Early Endocytic Vesicles in Mouse Liver

Nath

Bananis

Sarkar

et al. 2007

MBoC

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Early endocytic vesicles loaded with Texas Red asialoorosomucoid were prepared from mouse liver. These vesicles bound to microtubules in vitro, and upon ATP addition, they moved bidirectionally, frequently undergoing fission into two daughter vesicles. There was no effect of vanadate (inhibitor of dynein) on motility, whereas 5 -adenylylimido-diphosphate (kinesin inhibitor) was highly inhibitory. Studies with specific antibodies confirmed that dynein was not associated with these vesicles and that Kif5B and the minus-end kinesin Kifc1 mediated their plus-and minus-end motility, respectively. More than 90% of vesicles associated with Kifc1 also contained Kif5B, and inhibition of Kifc1 with antibody resulted in enhancement of plus-end-directed motility. There was reduced vesicle fission when either Kifc1 or Kif5B activity was inhibited by antibody, indicating that the opposing forces resulting from activity of both motors are required for fission to occur. Immunoprecipitation of native Kif5B by FLAG antibody after expression of FLAG-Kifc1 in 293T cells indicates that these two motors can interact with each other. Whether they interact directly or through a complex of potential regulatory proteins will need to be clarified in future studies. However, the present study shows that coordinated activity of these kinesins is essential for motility and processing of early endocytic vesicles. INTRODUCTIONReceptor-mediated endocytosis is a process in which ligands bind to specific cell surface receptors and internalize via clathrin-coated pits. After internalization the clathrin coat is released and uncoated vesicles mature into early endosomes (Mellman, 1996;Mukherjee et al., 1997;Marsh and McMahon, 1999;Higgins and McMahon, 2002;Perrais and Merrifield, 2005). After acidification of early endosomes, ligands such as asialoorosomucoid (ASOR) that are destined for lysosomes dissociate from their receptors (Harford et al., 1983a,b), and a series of fission events results in their segregation into separate daughter vesicles (Wolkoff et al., 1984;Mellman, 1996;Mukherjee et al., 1997;Murray and Wolkoff, 2003). The resulting ligand-enriched late endosomes traffic to lysosomes for degradation, whereas the receptor-containing daughter endosomes traffic back to the cell surface where receptor is reused (Harford et al., 1983a;Wolkoff et al., 1984;Mukherjee et al., 1997). Previous studies indicated that this segregation event requires an intact microtubule cytoskeleton (Goltz et al., 1992;Novikoff et al., 1996;Murray et al., 2000;Bananis et al., 2003Bananis et al., , 2004. In more recent studies, microtubule-based endosome motility and segregation were reconstituted in vitro using fluorescent early endosomes prepared from rat liver 5 min after portal venous injection of Texas Red-labeled ASOR, a substrate for the hepatocytespecific asialoglycoprotein receptor (ASGPR) (Bananis et al., , 2003(Bananis et al., , 2004Murray et al., 2000;Murray and Wolkoff, 2003). On addition of ATP to the in vitro assay, these vesicles moved bidirectionall...

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Section: Discussionsupporting

confidence: 93%

Kif5B and Kifc1 Interact and Are Required for Motility and Fission of Early Endocytic Vesicles in Mouse Liver

Nath

Bananis

Sarkar

et al. 2007

MBoC

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“…To assess the ability of CK2 to phosphorylate a molecular motor for FAT, we evaluated the phosphorylation status of squid kinesin-1 after oA␤ perfusion using the 63-90 monoclonal antibody against KLCs (38). The 63-90 antibody recognizes dephosphorylated KLCs (22,23).…”

Section: Perfusion Of Oa␤ Increased Phosphorylation Of Squid Kinesin-mentioning

confidence: 99%

Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta

Pigino

Morfini

Atagi

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

191

172

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The pathological mechanism by which A␤ causes neuronal dysfunction and death remains largely unknown. Deficiencies in fast axonal transport (FAT) were suggested to play a crucial role in neuronal dysfunction and loss for a diverse set of dying back neuropathologies including Alzheimer's disease (AD), but the molecular basis for pathological changes in FAT were undetermined. Recent findings indicate that soluble intracellular oligomeric A␤ (oA␤) species may play a critical role in AD pathology. Real-time analysis of vesicle mobility in isolated axoplasms perfused with oA␤ showed bidirectional axonal transport inhibition as a consequence of endogenous casein kinase 2 (CK2) activation. Conversely, neither unaggregated amyloid beta nor fibrillar amyloid beta affected FAT. Inhibition of FAT by oA␤ was prevented by two specific pharmacological inhibitors of CK2, as well as by competition with a CK2 substrate peptide. Furthermore, perfusion of axoplasms with active CK2 mimics the inhibitory effects of oA␤ on FAT. Both oA␤ and CK2 treatment of axoplasm led to increased phosphorylation of kinesin-1 light chains and subsequent release of kinesin from its cargoes. Therefore pharmacological modulation of CK2 activity may represent a promising target for therapeutic intervention in AD.Alzheimer's disease ͉ Axonal transport ͉ Beta amyloid oligomer ͉ CK2 ͉ Kinesin T he complex functional changes and histopathology of Alzheimer disease (AD) make it one of the most challenging disorders faced by modern medicine. Histopathological hallmarks of AD include distinctive extracellular and intracellular aggregates of amyloid beta (A␤) and tau (1, 2). Synaptic dysfunction and axonopathy appear to be the earliest lesions in AD as corroborated by reduced immunoreactivity of synaptophysin and other synaptic markers in terminal fields of brain-affected areas (3). AD-affected neurons appear to die eventually as a consequence of synaptic dysfunction and loss, following a typical dying-back pattern of neuronal degeneration.The amyloid hypothesis (4), a dominant concept in AD research for many years, has been revised in recent years to include the notion that smaller soluble A␤ aggregates may play an early and significant role in AD. Soluble oligomers of A␤ (oA␤) have been shown to be neurotoxic both in vivo and in vitro (5-7) as well as altering synaptic structure and function (8). Moreover, oA␤ levels correlate with impairments in cognitive function, learning, and memory (9, 10), but the molecular basis of these effects are uncertain. Intracellular A␤ was first described by Wertkin et al. (11). Immunogold electron microscopy showed that intraneuronal A␤ is pre-and postsynaptically enriched in both AD human brain and AD transgenic animal models in association with dystrophic neurites and abnormal synaptic morphology (12)(13)(14). Spatial and temporal analyses of intraneuronal oA␤ accumulation show that it precedes plaque formation in both AD animal models and Down's syndrome patients, suggesting that oA␤ is an early intracellular toxic agent i...

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“…In Drosophila KLC mutants, large aggregates ("organelle jams") accumulate in axons, resulting from a block in axonal transport (9). Also, anti-KLC antibodies inhibit fast axonal transport without affecting microtubule binding or ATPase activity in vitro and cause release of purified membrane vesicles from kinesin (14). Rat KLC1b binds via its C terminus to mitochondria in cultured CV1 cells and human skin fibroblasts (13).…”

Section: Discussionmentioning

confidence: 99%

“…Their role in KLC function may be similar. The highly variable C-terminal region of KLCs has been proposed to bind cargo such as organelles and macromolecules; this is supported by direct binding studies in which rat KLC1b was shown to bind to mitochondria (13) and by the use of specific antibodies that bind to KLC and block its interaction with organelles (14). As mentioned, KLCs may also regulate KHC and keep it in an inactive state by preventing the active conformation of KHC (11).…”

mentioning

confidence: 87%

“…KLCs have been suggested to bind cargo and to regulate the activity of KHCs. Clues as to possible functions and the domains involved came from sequence comparisons and genetic analysis of KLCs that were cloned from Caenorhabditis elegans (6), sea urchin (7), squid (8), Drosophila (9,10), and mammals (11)(12)(13)(14)(15)(16). In mouse, KLC1, a largely neuronal form of KLC, and the ubiquitous KLC2 have been identified (12).…”

mentioning

confidence: 99%

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Association of Kinesin Light Chain with Outer Dense Fibers in a Microtubule-independent Fashion

Bhullar

Zhang

Junco

et al. 2003

Journal of Biological Chemistry

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Conventional kinesin I motor molecules are heterotetramers consisting of two kinesin light chains (KLCs) and two kinesin heavy chains. The interaction between the heavy and light chains is mediated by the KLC heptad repeat (HR), a leucine zipper-like motif. Kinesins bind to microtubules and are involved in various cellular functions, including transport and cell division. We recently isolated a novel KLC gene, klc3. klc3 is the only known KLC expressed in post-meiotic male germ cells. A monoclonal anti-KLC3 antibody was developed that, in immunoelectron microscopy, detects KLC3 protein associated with outer dense fibers (ODFs), unique structural components of sperm tails. No significant binding of KLC3 with microtubules was observed with this monoclonal antibody. In vitro experiments showed that KLC3-ODF binding occurred in the absence of kinesin heavy chains or microtubules and required the KLC3 HR. ODF1, a major ODF protein, was identified as the KLC3 binding partner. The ODF1 leucine zipper and the KLC3 HR mediated the interaction. These results identify and characterize a novel interaction between a KLC and a non-microtubule macromolecular structure and suggest that KLC3 could play a microtubule-independent role during formation of sperm tails.

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Immunochemical analysis of kinesin light chain function.

Cited by 132 publications

References 60 publications

Kif5B and Kifc1 Interact and Are Required for Motility and Fission of Early Endocytic Vesicles in Mouse Liver

Kif5B and Kifc1 Interact and Are Required for Motility and Fission of Early Endocytic Vesicles in Mouse Liver

Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta

Association of Kinesin Light Chain with Outer Dense Fibers in a Microtubule-independent Fashion

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