scholarly journals Clonal Tests of Conventional Kinesin Function during Cell Proliferation and Differentiation

2000 ◽  
Vol 11 (4) ◽  
pp. 1329-1343 ◽  
Author(s):  
Robert P. Brendza ◽  
Kathy B. Sheehan ◽  
F.R. Turner ◽  
William M. Saxton
Keyword(s):  
Cell Proliferation ◽  
Endoplasmic Reticulum ◽  
Heavy Chain ◽  
Larval Instar ◽  
Vesicle Transport ◽  
Ultrastructural Changes ◽  
Chain Gene ◽  
Cell Periphery ◽  
Kinesin Heavy Chain ◽  
Conventional Kinesin

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.

scholarly journals Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent

2006 ◽  
Vol 173 (4) ◽  
pp. 545-557 ◽  
Author(s):  
Elizabeth E. Glater ◽  
Laura J. Megeath ◽  
R. Steven Stowers ◽  
Thomas L. Schwarz
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Adaptor Protein ◽  
Direct Interaction ◽  
Anterograde Transport ◽  
Kinesin Light Chain ◽  
Energy Demands ◽  
Kinesin Heavy Chain ◽  
Conventional Kinesin ◽  
Dependent Transport

Mitochondria are distributed within cells to match local energy demands. We report that the microtubule-dependent transport of mitochondria depends on the ability of milton to act as an adaptor protein that can recruit the heavy chain of conventional kinesin-1 (kinesin heavy chain [KHC]) to mitochondria. Biochemical and genetic evidence demonstrate that kinesin recruitment and mitochondrial transport are independent of kinesin light chain (KLC); KLC antagonizes milton's association with KHC and is absent from milton–KHC complexes, and mitochondria are present in klc −/− photoreceptor axons. The recruitment of KHC to mitochondria is, in part, determined by the NH2 terminus–splicing variant of milton. A direct interaction occurs between milton and miro, which is a mitochondrial Rho-like GTPase, and this interaction can influence the recruitment of milton to mitochondria. Thus, milton and miro are likely to form an essential protein complex that links KHC to mitochondria for light chain–independent, anterograde transport of mitochondria.

scholarly journals Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells

1992 ◽  
Vol 117 (6) ◽  
pp. 1263-1275 ◽  
Author(s):  
F Navone ◽  
J Niclas ◽  
N Hom-Booher ◽  
L Sparks ◽  
HD Bernstein ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Translational Regulation ◽  
Coiled Coil ◽  
Gene Interaction ◽  
Cloning And Expression ◽  
Upstream Open Reading Frame ◽  
Chain Gene ◽  
Terminal Domain ◽  
Kinesin Heavy Chain ◽  
Cytoplasmic Microtubules

To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOH-terminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesin-like protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with translational regulation in certain mRNAs. After transient expression in CV-1 cells, the kinesin heavy chain showed both a diffuse distribution and a filamentous staining pattern that coaligned with microtubules but not vimentin intermediate filaments. Altering the number and distribution of microtubules with taxol or nocodazole produced corresponding changes in the localization of the expressed kinesin heavy chain. The expressed NH2-terminal motor and the COOH-terminal tail domains, but not the alpha-helical coiled coil rod domain, also colocalized with microtubules. The finding that both the kinesin motor and tail domains can interact with cytoplasmic microtubules raises the possibility that kinesin could crossbridge and induce sliding between microtubules under certain circumstances.

scholarly journals Kinesin- and Myosin-driven Steps of Vesicle Recruitment for Ca2+-regulated Exocytosis

1997 ◽  
Vol 138 (5) ◽  
pp. 999-1008 ◽  
Author(s):  
Guo-Qiang Bi ◽  
Robert L. Morris ◽  
Guochun Liao ◽  
Janet M. Alderton ◽  
Jonathan M. Scholey ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Membrane Trafficking ◽  
Motor Proteins ◽  
Slow Phase ◽  
Cell Periphery ◽  
Tail Domain ◽  
Atpase Inhibitor ◽  
Intact Cells ◽  
Regulated Exocytosis ◽  
Kinesin Heavy Chain

Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca2+-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca2+/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin– dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca2+-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain.

scholarly journals Targeted Disruption of Mouse Conventional Kinesin Heavy Chain kif5B, Results in Abnormal Perinuclear Clustering of Mitochondria

Cell ◽  
1998 ◽  
Vol 93 (7) ◽  
pp. 1147-1158 ◽  
Author(s):  
Yosuke Tanaka ◽  
Yoshimitsu Kanai ◽  
Yasushi Okada ◽  
Shigenori Nonaka ◽  
Sen Takeda ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Targeted Disruption ◽  
Kinesin Heavy Chain ◽  
Conventional Kinesin

scholarly journals Evidence that the stalk of Drosophila kinesin heavy chain is an alpha-helical coiled coil.

1992 ◽  
Vol 116 (4) ◽  
pp. 957-965 ◽  
Author(s):  
M de Cuevas ◽  
T Tao ◽  
L S Goldstein
Keyword(s):  
Circular Dichroism ◽  
Heavy Chain ◽  
Coiled Coil ◽  
Chain Gene ◽  
Tail Domain ◽  
Major Transitions ◽  
Amino Terminal ◽  
Kinesin Heavy Chain ◽  
Stalk Domain ◽  
Circular Dichroïsm

Kinesin is a mechanochemical enzyme composed of three distinct domains: a globular head domain, a rodlike stalk domain, and a small globular tail domain. The stalk domain has sequence features characteristic of alpha-helical coiled coils. To gain insight into the structure of the kinesin stalk, we expressed it from a segment of the Drosophila melanogaster kinesin heavy chain gene and purified it from Escherichia coli. When observed by EM, this protein formed a rodlike structure 40-55 nm long that was occasionally bent at a hingelike region near the middle of the molecule. An additional EM study and a chemical cross-linking study showed that this protein forms a parallel dimer and that the two chains are in register. Finally, using circular dichroism spectroscopy, we showed that this protein is approximately 55-60% alpha-helical in physiological aqueous solution at 25 degrees C, and approximately 85-90% alpha-helical at 4 degrees C. From these results, we conclude that the stalk of kinesin heavy chain forms an alpha-helical coiled coil structure. The temperature dependence of the circular dichroism signal has two major transitions, at 25-30 degrees C and at 45-50 degrees C, which suggests that a portion of the alpha-helical structure in the stalk is less stable than the rest. By producing the amino-terminal (coil 1) and carboxy-terminal (coil 2) halves of the stalk separately in E. coli, we showed that the region that melts below 30 degrees C lies within coil 1, while the majority of coil 2 melts above 45 degrees C. We suggest that this difference in stability may play a role in the force-generating mechanism or regulation of kinesin.

scholarly journals Defective Kinesin Heavy Chain Behavior in Mouse Kinesin Light Chain Mutants

1999 ◽  
Vol 146 (6) ◽  
pp. 1277-1288 ◽  
Author(s):  
Amena Rahman ◽  
Adeela Kamal ◽  
Elizabeth A. Roberts ◽  
Lawrence S.B. Goldstein
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Mutant Mice ◽  
Kinesin Light Chain ◽  
Kinesin Heavy Chain ◽  
Neuronal Tissues ◽  
Biochemical Analyses ◽  
Conventional Kinesin

Conventional kinesin, kinesin-I, is a heterotetramer of two kinesin heavy chain (KHC) subunits (KIF5A, KIF5B, or KIF5C) and two kinesin light chain (KLC) subunits. While KHC contains the motor activity, the role of KLC remains unknown. It has been suggested that KLC is involved in either modulation of KHC activity or in cargo binding. Previously, we characterized KLC genes in mouse (Rahman, A., D.S. Friedman, and L.S. Goldstein. 1998. J. Biol. Chem. 273:15395–15403). Of the two characterized gene products, KLC1 was predominant in neuronal tissues, whereas KLC2 showed a more ubiquitous pattern of expression. To define the in vivo role of KLC, we generated KLC1 gene-targeted mice. Removal of functional KLC1 resulted in significantly smaller mutant mice that also exhibited pronounced motor disabilities. Biochemical analyses demonstrated that KLC1 mutant mice have a pool of KIF5A not associated with any known KLC subunit. Immunofluorescence studies of sensory and motor neuron cell bodies in KLC1 mutants revealed that KIF5A colocalized aberrantly with the peripheral cis-Golgi marker giantin in mutant cells. Striking changes and aberrant colocalization were also observed in the intracellular distribution of KIF5B and β′-COP, a component of COP1 coatomer. Taken together, these data best support models that suggest that KLC1 is essential for proper KHC activation or targeting.

scholarly journals Abnormal neurofilament transport caused by targeted disruption of neuronal kinesin heavy chain KIF5A

2003 ◽  
Vol 161 (1) ◽  
pp. 55-66 ◽  
Author(s):  
Chun-Hong Xia ◽  
Elizabeth A. Roberts ◽  
Lu-Shiun Her ◽  
Xinran Liu ◽  
David S. Williams ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Heavy Chain ◽  
Molecular Motor ◽  
Slow Axonal Transport ◽  
Targeted Disruption ◽  
Age Dependent ◽  
Kinesin Heavy Chain ◽  
Conventional Kinesin ◽  
Hind Limb Paralysis ◽  
Peripheral Sensory

To test the hypothesis that fast anterograde molecular motor proteins power the slow axonal transport of neurofilaments (NFs), we used homologous recombination to generate mice lacking the neuronal-specific conventional kinesin heavy chain, KIF5A. Because null KIF5A mutants die immediately after birth, a synapsin-promoted Cre-recombinase transgene was used to direct inactivation of KIF5A in neurons postnatally. Three fourths of such mutant mice exhibited seizures and death at around 3 wk of age; the remaining animals survived to 3 mo or longer. In young mutant animals, fast axonal transport appeared to be intact, but NF-H, as well as NF-M and NF-L, accumulated in the cell bodies of peripheral sensory neurons accompanied by a reduction in sensory axon caliber. Older animals also developed age-dependent sensory neuron degeneration, an accumulation of NF subunits in cell bodies and a reduction in axons, loss of large caliber axons, and hind limb paralysis. These data support the hypothesis that a conventional kinesin plays a role in the microtubule-dependent slow axonal transport of at least one cargo, the NF proteins.

Ultrastructural Changes Associated with Alcoholic Hyalin in Hepatocytes and Biliary Epithelial Cells

1971 ◽  
Vol 29 ◽  
pp. 572-573
Author(s):  
Odell T. Minick ◽  
Hidejiro Yokoo ◽  
Fawzia Batti
Keyword(s):  
Endoplasmic Reticulum ◽  
Epithelial Cells ◽  
Liver Biopsy ◽  
Alcoholic Hepatitis ◽  
Ultrastructural Changes ◽  
Main Mass ◽  
Biliary Epithelial Cells ◽  
Biopsy Specimens ◽  
Alcoholic Hyalin

To learn more of the nature and origin of alcoholic hyalin (AH), 15 liver biopsy specimens from patients with alcoholic hepatitis were studied in detail.AH was found not only in hepatocytes but also in ductular cells (Figs. 1 and 2), although in the latter location only rarely. The bulk of AH consisted of a randomly oriented network of closely packed filaments measuring about 150 Å in width. Bundles of filaments smaller in diameter (40-90 Å) were observed along the periphery of the main mass (Fig. 1), often surrounding it in a rim-like fashion. Fine filaments were also found close to the nucleus in both hepatocytes and biliary epithelial cells, the latter even though characteristic AH was not present (Figs. 3 and 4). Dispersed among the larger filaments were glycogen, RNA particles and profiles of endoplasmic reticulum. Dilated cisternae of endoplasmic reticulum were often conspicuous around the periphery of the AH mass. A limiting membrane was not observed.

Ultrastructural Changes of Smoooth Endoplasmic Reticulum in the Retinal Pigment Epithelium by Choline Chloride

1972 ◽  
Vol 30 ◽  
pp. 58-59
Author(s):  
Kazushige Hirosawa ◽  
Eichi Yamada
Keyword(s):  
Endoplasmic Reticulum ◽  
Epithelial Cell ◽  
Smooth Endoplasmic Reticulum ◽  
Pigment Epithelium ◽  
Choline Chloride ◽  
Retinal Pigment ◽  
Ultrastructural Changes ◽  
Lower Vertebrate ◽  
Visual Cells ◽  
Pigment Epithelial

The pigment epithelium is located between the choriocapillary and the visual cells. The pigment epithelial cell is characterized by a large amount of the smooth endoplasmic reticulum (SER) in its cytoplasm. In addition, the pigment epithelial cell of some lower vertebrate has myeloid body as a specialized form of the SER. Generally, SER is supposed to work in the lipid metabolism. However, the functions of abundant SER and myeloid body in the pigment epithelial cell are still in question. This paper reports an attempt, to depict the functions of these organelles in the frog retina by administering one of phospholipid precursors.

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