scholarly journals Interactions of LC8 with N-Terminal Segments of the Intermediate Chain of Cytoplasmic Dynein

2003 ◽  
Vol 3 ◽  
pp. 647-654 ◽  
Author(s):  
Afua Nyarko ◽  
Michael Hare ◽  
Moses Makokha ◽  
Elisar Barbar
Keyword(s):  
Limited Proteolysis ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Light Chains ◽  
Ordered Structure ◽  
Dynein Motor ◽  
Dynein Light Chains ◽  
Significant Conformational Change ◽  
Intermediate Chain

LC8, a highly conserved 10-kDa light chain, and IC74, a 74-kDa intermediate chain, are presumed to promote the assembly of the cytoplasmic dynein motor protein complex and to be engaged in the controlled binding and release of cargo. The interactions of LC8 from Drosophila melanogaster with constructs of IC74 were characterized in vitro by affinity methods, limited proteolysis, and circular dichroism spectroscopy. Previously, we have performed limited proteolysis on the N-terminal domain of IC74, IC(1-289), when free and when bound to dynein light chains LC8 and Tctex-1[1]. We have also shown that upon addition of LC8, IC(1-289) undergoes a significant conformational change from a largely unfolded to a more ordered structure. The purpose of the work presented here is to determine whether residues 1-30 in IC74, predicted to be in a coiled coil, are involved in the stabilization of the protein upon binding to LC8. Constructs of IC74, IC(1-143), and IC(30-143) that include the LC8 binding site but with and without the first 30 residues were prepared, and their binding and protection patterns were compared to our previous results for IC(1-289). The results suggest that coiled coil residues 1-30 are not responsible for the increase in structure we observe when IC(1-289) binds to LC8.

scholarly journals Interactions of Cytoplasmic Dynein Light Chains Tctex-1 and LC8 with the Intermediate Chain IC74†

Biochemistry ◽  
2002 ◽  
Vol 41 (13) ◽  
pp. 4302-4311 ◽  
Author(s):  
Moses Makokha ◽  
Michael Hare ◽  
Mingang Li ◽  
Thomas Hays ◽  
Elisar Barbar
Keyword(s):  
Cytoplasmic Dynein ◽  
Light Chains ◽  
Dynein Light Chains ◽  
Intermediate Chain

scholarly journals Stable tug-of-war between kinesin-1 and cytoplasmic dynein upon different ATP and roadblock concentrations

2020 ◽  
Author(s):  
Gina A. Monzon ◽  
Lara Scharrel ◽  
Ashwin DSouza ◽  
Ludger Santen ◽  
Stefan Diez
Keyword(s):  
Cytoplasmic Dynein ◽  
Gliding Motility ◽  
Force Balance ◽  
Stochastic Simulations ◽  
Transport Systems ◽  
Organelle Transport ◽  
Atp Concentration ◽  
Dynein Motor ◽  
Bidirectional Movement

ABSTRACTThe maintenance of intracellular processes like organelle transport and cell division depend on bidirectional movement along microtubules. These processes typically require kinesin and dynein motor proteins which move with opposite directionality. Because both types of motors are often simultaneously bound to the cargo, regulatory mechanisms are required to ensure controlled directional transport. Recently, it has been shown that parameters like mechanical motor activation, ATP concentration and roadblocks on the microtubule surface differentially influence the activity of kinesin and dynein motors in distinct manners. However, how these parameters affect bidirectional transport systems has not been studied. Here, we investigate the regulatory influence of these three parameter using in vitro gliding motility assays and stochastic simulations. We find that the number of active kinesin and dynein motors determines the transport direction and velocity, but that variations in ATP concentration and roadblock density have no significant effect. Thus, factors influencing the force balance between opposite motors appear to be important, whereas the detailed stepping kinetics and bypassing capabilities of the motors have only little effect.

scholarly journals Dynactin Is Required for Microtubule Anchoring at Centrosomes

1999 ◽  
Vol 147 (2) ◽  
pp. 321-334 ◽  
Author(s):  
N.J. Quintyne ◽  
S.R. Gill ◽  
D.M. Eckley ◽  
C.L. Crego ◽  
D.A. Compton ◽  
...  
Keyword(s):  
Mitotic Cell ◽  
Cytoplasmic Dynein ◽  
Microtubule Organization ◽  
Cultured Fibroblasts ◽  
Cell Extracts ◽  
Dynein Motor ◽  
Mitotic Spindle Assembly ◽  
Dynactin Complex

The multiprotein complex, dynactin, is an integral part of the cytoplasmic dynein motor and is required for dynein-based motility in vitro and in vivo. In living cells, perturbation of the dynein–dynactin interaction profoundly blocks mitotic spindle assembly, and inhibition or depletion of dynein or dynactin from meiotic or mitotic cell extracts prevents microtubules from focusing into spindles. In interphase cells, perturbation of the dynein–dynactin complex is correlated with an inhibition of ER-to-Golgi movement and reorganization of the Golgi apparatus and the endosome–lysosome system, but the effects on microtubule organization have not previously been defined. To explore this question, we overexpressed a variety of dynactin subunits in cultured fibroblasts. Subunits implicated in dynein binding have effects on both microtubule organization and centrosome integrity. Microtubules are reorganized into unfocused arrays. The pericentriolar components, γ tubulin and dynactin, are lost from centrosomes, but pericentrin localization persists. Microtubule nucleation from centrosomes proceeds relatively normally, but microtubules become disorganized soon thereafter. Overexpression of some, but not all, dynactin subunits also affects endomembrane localization. These data indicate that dynein and dynactin play important roles in microtubule organization at centrosomes in fibroblastic cells and provide new insights into dynactin–cargo interactions.

scholarly journals Somatic CRISPR–Cas9-induced mutations reveal roles of embryonically essential dynein chains in Caenorhabditis elegans cilia

2015 ◽  
Vol 208 (6) ◽  
pp. 683-692 ◽  
Author(s):  
Wenjing Li ◽  
Peishan Yi ◽  
Guangshuo Ou
Keyword(s):  
Caenorhabditis Elegans ◽  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Regulatory Mechanisms ◽  
Light Chains ◽  
Induced Mutations ◽  
Functional Studies ◽  
Cilium Formation ◽  
Dynein Intermediate Chain ◽  
Intermediate Chain

Cilium formation and maintenance require intraflagellar transport (IFT). Although much is known about kinesin-2–driven anterograde IFT, the composition and regulation of retrograde IFT-specific dynein remain elusive. Components of cytoplasmic dynein may participate in IFT; however, their essential roles in cell division preclude functional studies in postmitotic cilia. Here, we report that inducible expression of the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system in Caenorhabditis elegans generated conditional mutations in IFT motors and particles, recapitulating ciliary defects in their null mutants. Using this method to bypass the embryonic requirement, we show the following: the dynein intermediate chain, light chain LC8, and lissencephaly-1 regulate retrograde IFT; the dynein light intermediate chain functions in dendrites and indirectly contributes to ciliogenesis; and the Tctex and Roadblock light chains are dispensable for cilium assembly. Furthermore, we demonstrate that these components undergo biphasic IFT with distinct transport frequencies and turnaround behaviors. Together, our results suggest that IFT–dynein and cytoplasmic dynein have unique compositions but also share components and regulatory mechanisms.

scholarly journals Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein.

1991 ◽  
Vol 115 (6) ◽  
pp. 1639-1650 ◽  
Author(s):  
S R Gill ◽  
T A Schroer ◽  
I Szilak ◽  
E R Steuer ◽  
M P Sheetz ◽  
...  
Keyword(s):  
Single Gene ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Protein Product ◽  
Structural Motif ◽  
Sequence Identity ◽  
Vesicle Movement ◽  
Dynactin Complex ◽  
Activator Complex

Although cytoplasmic dynein is known to attach to microtubules and translocate toward their minus ends, dynein's ability to serve in vitro as a minus end-directed transporter of membranous organelles depends on additional soluble factors. We show here that a approximately 20S polypeptide complex (referred to as Activator I; Schroer, T. A., and M.P. Sheetz. 1991a. J. Cell Biol. 115:1309-1318.) stimulates dynein-mediated vesicle transport. A major component of the activator complex is a doublet of 150-kD polypeptides for which we propose the name dynactin (for dynein activator). The 20S dynactin complex is required for in vitro vesicle motility since depletion of it with a mAb to dynactin eliminates vesicle movement. Cloning of a brain specific isoform of dynactin from chicken reveals a 1,053 amino acid polypeptide composed of two coiled-coil alpha-helical domains interrupted by a spacer. Both this structural motif and the underlying primary sequence are highly conserved in vertebrates with 85% sequence identity within a central 1,000-residue domain of the chicken and rat proteins. As abundant as dynein, dynactin is ubiquitously expressed and appears to be encoded by a single gene that yields at least three alternative isoforms. The probable homologue in Drosophila is the gene Glued, whose protein product shares 50% sequence identity with vertebrate dynactin and whose function is essential for viability of most (and perhaps all) cells in the organism.

scholarly journals Cytoplasmic Dynein Light Intermediate Chain Is Required for Discrete Aspects of Mitosis in Caenorhabditis elegans

2001 ◽  
Vol 12 (10) ◽  
pp. 2921-2933 ◽  
Author(s):  
John H. Yoder ◽  
Min Han
Keyword(s):  
Caenorhabditis Elegans ◽  
Nucleotide Binding ◽  
Cytoplasmic Dynein ◽  
Meiotic Spindle ◽  
Phenotypic Characterization ◽  
Loss Of Function ◽  
Dynein Motor ◽  
Centrosome Separation ◽  
Transporter Family ◽  
Intermediate Chain

We describe phenotypic characterization of dli-1, the Caenorhabditis elegans homolog of cytoplasmic dynein light intermediate chain (LIC), a subunit of the cytoplasmic dynein motor complex. Animals homozygous for loss-of-function mutations indli-1 exhibit stochastic failed divisions in late larval cell lineages, resulting in zygotic sterility. dli-1 is required for dynein function during mitosis. Depletion of thedli-1 gene product through RNA-mediated gene interference (RNAi) reveals an early embryonic requirement. One-celldli-1(RNAi) embryos exhibit failed cell division attempts, resulting from a variety of mitotic defects. Specifically, pronuclear migration, centrosome separation, and centrosome association with the male pronuclear envelope are defective indli-1(RNAi) embryos. Meiotic spindle formation, however, is not affected in these embryos. DLI-1, like its vertebrate homologs, contains a putative nucleotide-binding domain similar to those found in the ATP-binding cassette transporter family of ATPases as well as other nucleotide-binding and -hydrolyzing proteins. Amino acid substitutions in a conserved lysine residue, known to be required for nucleotide binding, confers complete rescue in a dli-1mutant background, indicating this is not an essential domain for DLI-1 function.

scholarly journals The Gene for the Intermediate Chain Subunit of Cytoplasmic Dynein Is Essential in Drosophila

Genetics ◽  
2002 ◽  
Vol 162 (3) ◽  
pp. 1211-1220 ◽  
Author(s):  
Kristin L M Boylan ◽  
Thomas S Hays
Keyword(s):  
Intracellular Transport ◽  
De Novo ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Temperature Sensitive ◽  
Wing Development ◽  
Dynein Motor ◽  
Dynein Intermediate Chain ◽  
Microtubule Motor ◽  
Intermediate Chain

Abstract The microtubule motor cytoplasmic dynein powers a variety of intracellular transport events that are essential for cellular and developmental processes. A current hypothesis is that the accessory subunits of the dynein complex are important for the specialization of cytoplasmic dynein function. In a genetic approach to understanding the range of dynein functions and the contribution of the different subunits to dynein motor function and regulation, we have identified mutations in the gene for the cytoplasmic dynein intermediate chain, Dic19C. We used a functional Dic transgene in a genetic screen to recover X-linked lethal mutations that require this transgene for viability. Three Dic mutations were identified and characterized. All three Dic alleles result in larval lethality, demonstrating that the intermediate chain serves an essential function in Drosophila. Like a deficiency that removes Dic19C, the Dic mutations dominantly enhance the rough eye phenotype of Glued1, a dominant mutation in the gene for the p150 subunit of the dynactin complex, a dynein activator. Additionally, we used complementation analysis to identify an existing mutation, shortwing (sw), as an allele of the dynein intermediate chain gene. Unlike the Dic alleles isolated de novo, shortwing is homozygous viable and exhibits recessive and temperature-sensitive defects in eye and wing development. These phenotypes are rescued by the wild-type Dic transgene, indicating that shortwing is a viable allele of the dynein intermediate chain gene and revealing a novel role for dynein function during wing development.

scholarly journals Dynein Intermediate Chain Mediated Dynein–Dynactin Interaction Is Required for Interphase Microtubule Organization and Centrosome Replication and Separation inDictyostelium

1999 ◽  
Vol 147 (6) ◽  
pp. 1261-1274 ◽  
Author(s):  
Shuo Ma ◽  
Leda Triviños-Lagos ◽  
Ralph Gräf ◽  
Rex L. Chisholm
Keyword(s):  
Heavy Chain ◽  
Physiological Role ◽  
Cytoplasmic Dynein ◽  
Wild Type ◽  
Microtubule Organization ◽  
Dynein Intermediate Chain ◽  
Organizing Center ◽  
Intermediate Chain

Cytoplasmic dynein intermediate chain (IC) mediates dynein–dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806–28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507–1516). To investigate the physiological role of IC and dynein–dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICΔC associated with dynactin but not with dynein heavy chain, whereas ICΔN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.

scholarly journals Lissencephaly-1 is a context-dependent regulator of the human dynein complex

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Janina Baumbach ◽  
Andal Murthy ◽  
Mark A McClintock ◽  
Carly I Dix ◽  
Ruta Zalyte ◽  
...  
Keyword(s):  
Complex Formation ◽  
Binding Protein ◽  
Cytoplasmic Dynein ◽  
Cargo Transport ◽  
Dynein Motor ◽  
Context Dependent ◽  
Shed Light ◽  
Dynamic Microtubule

The cytoplasmic dynein-1 (dynein) motor plays a central role in microtubule organisation and cargo transport. These functions are spatially regulated by association of dynein and its accessory complex dynactin with dynamic microtubule plus ends. Here, we elucidate in vitro the roles of dynactin, end-binding protein-1 (EB1) and Lissencephaly-1 (LIS1) in the interaction of end tracking and minus end-directed human dynein complexes with these sites. LIS1 promotes dynactin-dependent tracking of dynein on both growing and shrinking plus ends. LIS1 also increases the frequency and velocity of processive dynein movements that are activated by complex formation with dynactin and a cargo adaptor. This stimulatory effect of LIS1 contrasts sharply with its documented ability to inhibit the activity of isolated dyneins. Collectively, our findings shed light on how mammalian dynein complexes associate with dynamic microtubules and help clarify how LIS1 promotes the plus-end localisation and cargo transport functions of dynein in vivo.

scholarly journals Polymerization pathway of mammalian nonmuscle myosin 2s

2018 ◽  
Vol 115 (30) ◽  
pp. E7101-E7108 ◽  
Author(s):  
Xiong Liu ◽  
Shi Shu ◽  
Edward D. Korn
Keyword(s):  
Coiled Coil ◽  
Polymerization Process ◽  
Light Chains ◽  
Biological Functions ◽  
Nonmuscle Myosin ◽  
Heavy Chains ◽  
Filament Dynamics ◽  
Bipolar Filament

The three mammalian nonmuscle myosin 2 (NM2) monomers, like all class 2 myosin monomers, are hexamers of two identical heavy (long) chains and two pairs of light (short) chains bound to the heavy chains. The heavy chains have an N-terminal globular motor domain (head) with actin-activated ATPase activity, a lever arm (neck) to which the two light chains bind, and a coiled-coil helical tail. Monomers polymerize into bipolar filaments, with globular heads at each end separated by a bare zone, by antiparallel association of their coiled-coil tails. NM2 filaments are highly dynamic in situ, frequently disassembling and reassembling at different locations within the cell where they are essential for multiple biological functions. Therefore, it is important to understand the mechanisms of filament polymerization and depolymerization. Monomers can exist in two states: folded and unfolded. It has been thought that unfolded monomers form antiparallel dimers that assemble into bipolar filaments. We now show that polymerization in vitro proceeds from folded monomers to folded antiparallel dimers to folded antiparallel tetramers that unfold forming antiparallel bipolar tetramers. Folded dimers and tetramers then associate with the unfolded tetramer and unfold, forming a mature bipolar filament consisting of multiple unfolded tetramers with an entwined bare zone. We also demonstrate that depolymerization is essentially the reverse of the polymerization process. These results will advance our understanding of NM2 filament dynamics in situ.

Sign in / Sign up

Export Citation Format

Share Document