scholarly journals Kinesin Light Chains Are Essential for Axonal Transport in Drosophila

1998 ◽  
Vol 141 (2) ◽  
pp. 443-454 ◽  
Author(s):  
Joseph G. Gindhart ◽  
Chand J. Desai ◽  
Sven Beushausen ◽  
Kai Zinn ◽  
Lawrence S.B. Goldstein
Keyword(s):  
Axonal Transport ◽  
Larval Instar ◽  
Cytoplasmic Dynein ◽  
Light Chains ◽  
Kinesin Light Chain ◽  
Heavy Chains ◽  
Segmental Nerve ◽  
Microtubule Motor ◽  
Function Blocks ◽  
Mutant Phenotypes

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 (Saxton et al. 1991. 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.

scholarly journals Release of Kinesin from Vesicles by hsc70 and Regulation of Fast Axonal Transport

2000 ◽  
Vol 11 (6) ◽  
pp. 2161-2173 ◽  
Author(s):  
Ming-Ying Tsai ◽  
Gerardo Morfini ◽  
Györgyi Szebenyi ◽  
Scott T. Brady
Keyword(s):  
Axonal Transport ◽  
Light Chain ◽  
Tandem Repeats ◽  
Light Chains ◽  
Fast Axonal Transport ◽  
Kinesin Light Chain ◽  
Highly Sensitive ◽  
Subcellular Domains ◽  
Buffer Composition ◽  
Membrane Bound

The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of eitherN-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent andN-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.

scholarly journals The light chains of kinesin-1 are autoinhibited

2016 ◽  
Vol 113 (9) ◽  
pp. 2418-2423 ◽  
Author(s):  
Yan Y. Yip ◽  
Stefano Pernigo ◽  
Anneri Sanger ◽  
Mengjia Xu ◽  
Maddy Parsons ◽  
...  
Keyword(s):  
Intramolecular Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Light Chains ◽  
Heavy Chains ◽  
Linear Peptide ◽  
Peptide Motifs ◽  
Microtubule Motor ◽  
Tetratricopeptide Repeat Domain ◽  
Cargo Binding

The light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLCTPR), they can recognize short linear peptide motifs found in many cargo proteins characterized by a central tryptophan flanked by aspartic/glutamic acid residues (W-acidic). Using a fluorescence resonance energy transfer biosensor in combination with X-ray crystallographic, biochemical, and biophysical approaches, we describe how an intramolecular interaction between the KLC2TPR domain and a conserved peptide motif within an unstructured region of the molecule, partly occludes the W-acidic binding site on the TPR domain. Cargo binding displaces this interaction, effecting a global conformational change in KLCs resulting in a more extended conformation. Thus, like the motor-bearing kinesin heavy chains, KLCs exist in a dynamic conformational state that is regulated by self-interaction and cargo binding. We propose a model by which, via this molecular switch, W-acidic cargo binding regulates the activity of the holoenzyme.

An isoform of kinesin light chain specific for the Golgi complex

2000 ◽  
Vol 113 (11) ◽  
pp. 2047-2054
Author(s):  
F.K. Gyoeva ◽  
E.M. Bybikova ◽  
A.A. Minin
Keyword(s):  
Golgi Complex ◽  
Light Chain ◽  
Cultured Cells ◽  
Light Chains ◽  
Western Blots ◽  
Kinesin Light Chain ◽  
Heavy Chains ◽  
Carboxyl Terminal ◽  
Kinesin Molecule ◽  
Conventional Kinesin

Conventional kinesin is a motor protein implicated in the transport of a variety of cytoplasmic organelles along microtubules. The kinesin molecule consists of two heavy chains with motor domains at their amino termini and two light chains, which, together with the carboxyl termini of the heavy chains, are proposed to mediate binding to cargoes. Since the light chains are represented by multiple isoforms diverging at their carboxyl termini they are presumed to specify kinesin targeting to organelles. Previously, we isolated five cDNAs, encoding hamster kinesin light chain isoforms, and found that one of them (B or C) preferentially associated with mitochondria. To obtain additional evidence proving the specific location of various kinesin light chain isoforms on organelles, we made an antibody against a 56 amino-acid sequence found at the carboxyl-terminal regions of the hamster D and E isoforms. By indirect immunofluorescence, this antibody specifically labeled the Golgi complex in cultured cells. In western blots of total cell homogenates, it recognized two close polypeptides, one of which co-purified with the Golgi membranes. Thus, the results of this and previous studies demonstrate that different kinesin light chains are associated with different organelles in cells.

scholarly journals An essential post-developmental role for Lis1 in mice

2017 ◽  
Author(s):  
Timothy J. Hines ◽  
Xu Gao ◽  
Subhshri Sahu ◽  
Meghann M. Lange ◽  
Jill R. Turner ◽  
...  
Keyword(s):  
Nervous System ◽  
Axonal Transport ◽  
Time Frame ◽  
Rapid Onset ◽  
Cytoplasmic Dynein ◽  
Vital Role ◽  
Adult Mice ◽  
Microtubule Motor ◽  
Actin Promoter ◽  
Axonal Dysfunction

ABSTRACTLIS1 mutations cause lissencephaly (LIS), a severe developmental brain malformation. Much less is known about its role in the mature nervous system. LIS1 regulates the microtubule motor cytoplasmic dynein 1 (dynein), and as LIS1 and dynein are both expressed in the adult nervous system, Lis1 could potentially regulate dynein-dependent processes such as axonal transport. We therefore knocked out Lis1 in adult mice using tamoxifen-induced, Cre-ER-mediated recombination. When an actin promoter was used to drive Cre-ER expression (Act-Cre-ER), heterozygous Lis1 KO caused no obvious change in viability or behavior, despite evidence of widespread recombination by a Cre reporter three weeks after tamoxifen exposure. In contrast, homozygous Lis1 KO caused the rapid onset of neurological symptoms in both male and female mice. One tamoxifen-dosing regimen caused prominent recombination in the midbrain/hindbrain, PNS, and cardiac/skeletal muscle within a week; these mice developed severe symptoms in that time frame and were euthanized. A different tamoxifen regimen resulted in delayed recombination in midbrain/hindbrain, but not in other tissues, and also delayed the onset of symptoms. This indicates that Lis1 loss in the midbrain/hindbrain causes the severe phenotype. In support of this, brainstem regions known to house cardiorespiratory centers showed signs of axonal dysfunction in KO animals. Transport defects, neurofilament alterations, and varicosities were observed in axons in cultured DRG neurons from KO animals. Because no symptoms were observed when a cardiac specific Cre-ER promoter was used, we propose a vital role for Lis1 in autonomic neurons and implicate defective axonal transport in the KO phenotype.SIGNIFICANCE STATEMENTMammalian Lis1 is best known for its role in brain development. Lis1 binds to and regulates the microtubule motor, cytoplasmic dynein. We show that Lis1 function is needed post-developmentally and provide evidence that loss of Lis1 in the hindbrain leads to death. The effect is dose dependent in mice, as loss of only one allele does not produce an overt phenotype. However, since LIS1 haploinsufficiency causes lissencephaly (LIS) in humans, our study raises the possibility that post-developmental axonal transport defects could contribute to worsening symptoms in children with LIS1 mutations. Our data are consistent with the hypothesis is that Lis1 regulates dynein-dependent axon transport in the mature nervous system.

scholarly journals Immunochemical analysis of kinesin light chain function.

1997 ◽  
Vol 8 (4) ◽  
pp. 675-689 ◽  
Author(s):  
D L Stenoien ◽  
S T Brady
Keyword(s):  
Axonal Transport ◽  
Light Chain ◽  
Membrane Vesicles ◽  
Retrograde Transport ◽  
Light Chains ◽  
Fast Axonal Transport ◽  
Kinesin Light Chain ◽  
Punctate Pattern ◽  
Conventional Kinesin

The kinesin heterotetramer consists of two heavy and two light chains. Kinesin light chains have been proposed to act in binding motor protein to cargo, but evidence for this has been indirect. A library of monoclonal antibodies directed against conserved epitopes throughout the kinesin light chain sequence were used to map light chain functional architecture and to assess physiological functions of these domains. Immunocytochemistry with all antibodies showed a punctate pattern that was detergent soluble. A monoclonal antibody (KLC-All) made against a highly conserved epitope in the tandem repeat domain of light chains inhibited fast axonal transport in isolated axoplasm by decreasing both the number and velocity of vesicles moving, whereas an antibody against a conserved amino terminus epitope had no effect. KLC-All was equally effective at inhibiting both anterograde and retrograde transport. Neither antibody inhibited microtubule-binding or ATPase activity in vitro. KLC-All was unique among antibodies tested in releasing kinesin from purified membrane vesicles, suggesting a mechanism of action for inhibition of axonal transport. These results provide further evidence that conventional kinesin is a motor for fast axonal transport and demonstrate that kinesin light chains play an important role in kinesin interaction with membranes.

scholarly journals The Interaction of Neurofilaments with the Microtubule Motor Cytoplasmic Dynein

2004 ◽  
Vol 15 (11) ◽  
pp. 5092-5100 ◽  
Author(s):  
Oliver I. Wagner ◽  
Jennifer Ascaño ◽  
Mariko Tokito ◽  
Jean-Francois Leterrier ◽  
Paul A. Janmey ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Specific Interaction ◽  
Cytoplasmic Dynein ◽  
Live Cells ◽  
Slow Axonal Transport ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microtubule Motors ◽  
Microtubule Motor ◽  
Physical Interactions

Neurofilaments are synthesized in the cell body of neurons and transported outward along the axon via slow axonal transport. Direct observation of neurofilaments trafficking in live cells suggests that the slow outward rate of transport is due to the net effects of anterograde and retrograde microtubule motors pulling in opposition. Previous studies have suggested that cytoplasmic dynein is required for efficient neurofilament transport. In this study, we examine the interaction of neurofilaments with cytoplasmic dynein. We used fluid tapping mode atomic force microscopy to visualize single neurofilaments, microtubules, dynein/dynactin, and physical interactions between these neuronal components. AFM images suggest that neurofilaments act as cargo for dynein, associating with the base of the motor complex. Yeast two-hybrid and affinity chromatography assays confirm this hypothesis, indicating that neurofilament subunit M binds directly to dynein IC. This interaction is blocked by monoclonal antibodies directed either to NF-M or to dynein. Together these data suggest that a specific interaction between neurofilament subunit M and cytoplasmic dynein is involved in the saltatory bidirectional motility of neurofilaments undergoing axonal transport in the neuron.

Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography

1988 ◽  
Vol 46 ◽  
pp. 156-157
Author(s):  
Donald A. Winkelmann
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Actin Filaments ◽  
Light Chains ◽  
Myosin Filament ◽  
Primary Role ◽  
Heavy Chains ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Globular Head

The primary role of the interaction of actin and myosin is the generation of force and motion as a direct consequence of the cyclic interaction of myosin crossbridges with actin filaments. Myosin is composed of six polypeptides: two heavy chains of molecular weight 220,000 daltons and two pairs of light chains of molecular weight 17,000-23,000. The C-terminal portions of the myosin heavy chains associate to form an α-helical coiled-coil rod which is responsible for myosin filament formation. The N-terminal portion of each heavy chain associates with two different light chains to form a globular head that binds actin and hydrolyses ATP. Myosin can be fragmented by limited proteolysis into several structural and functional domains. It has recently been demonstrated using an in vitro movement assay that the globular head domain, subfragment-1, is sufficient to cause sliding movement of actin filaments.The discovery of conditions for crystallization of the myosin subfragment-1 (S1) has led to a systematic analysis of S1 structure by x-ray crystallography and electron microscopy. Image analysis of electron micrographs of thin sections of small S1 crystals has been used to determine the structure of S1 in the crystal lattice.

scholarly journals Spastic Paraplegia Mutation N256S in the Neuronal Microtubule Motor KIF5A Disrupts Axonal Transport in a Drosophila HSP Model

PLoS Genetics ◽  
2012 ◽  
Vol 8 (11) ◽  
pp. e1003066 ◽  
Author(s):  
Petra Füger ◽  
Vrinda Sreekumar ◽  
Rebecca Schüle ◽  
Jeannine V. Kern ◽  
Doychin T. Stanchev ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Spastic Paraplegia ◽  
Microtubule Motor

scholarly journals Identification and developmental regulation of a neuron-specific subunit of cytoplasmic dynein.

1996 ◽  
Vol 7 (2) ◽  
pp. 331-343 ◽  
Author(s):  
K K Pfister ◽  
M W Salata ◽  
J F Dillman ◽  
E Torre ◽  
R J Lye
Keyword(s):  
Axonal Transport ◽  
Developmental Regulation ◽  
Cytoplasmic Dynein ◽  
Retrograde Axonal Transport ◽  
Protein Isoforms ◽  
Cultured Neurons ◽  
Developmentally Regulated ◽  
Time Period ◽  
Intermediate Chains

Cytoplasmic dynein is the microtubule minus-end-directed motor for the retrograde axonal transport of membranous organelles. Because of its similarity to the intermediate chains of flagellar dynein, the 74-kDa intermediate chain (IC74) subunit of dynein is thought to be involved in binding dynein to its membranous organelle cargo. Previously, we identified six isoforms of the IC74 cytoplasmic dynein subunit in the brain. We further demonstrated that cultured glia and neurons expressed different dynein IC74 isoforms and phospho-isoforms. Two isoforms were observed when dynein from glia was analyzed. When dynein from cultured neurons was analyzed, six IC74 isoforms were observed, although the relative amounts of the dynein isoforms from cultured neurons differed from those found in dynein from brain. To better understand the role of the neuronal IC74 isoforms and identify neuron-specific IC74 dynein subunits, the expression of the IC74 protein isoforms and mRNAs of various tissues were compared. As a result of this comparison, the identity of each of the isoform spots observed on two-dimensional gels was correlated with the products of each of the IC74 mRNAs. We also found that between the fifteenth day of gestation (E15) and the fifth day after birth (P5), the relative expression of the IC74 protein isoforms changes, demonstrating that the expression of IC74 isoforms is developmentally regulated in brain. During this time period, there is relatively little change in the abundance of the various IC74 mRNAs. The E15 to P5 time period is one of rapid process extension and initial pattern formation in the rat brain. This result indicates that the changes in neuronal IC74 isoforms coincide with neuronal differentiation, in particular the extension of processes. This suggests a role for the neuronal IC74 isoforms in the establishment or regulation of retrograde axonal transport.

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