Biochemical and Molecular Analysis of the Mammalian Cytoplasmic Dynein Intermediate Chain

Brill, Louis B.; Pfister, Karin

doi:10.1006/meth.2000.1083

Cited by 15 publications

(19 citation statements)

References 45 publications

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“…The native cytoplasmic dynein complex was purified by immuno-affinity chromatography from rat brain and other rat tissues using the 74.1 monoclonal antibody as described previously [Dillman and Pfister, 1994;Brill and Pfister, 2000]. The dynein light chains were resolved on 4 -16% polyacrylamide gels (stabilized with glycerol) and identified by staining with Coomassie blue or silver (Silver Stain Plus, BioRad).…”

Section: Methodsmentioning

confidence: 99%

Light chains of mammalian cytoplasmic dynein: identification and characterization of a family of LC8 light chains

Wilson

Salata

Susalka

et al. 2001

Cell Motil. Cytoskeleton

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Cytoplasmic dynein is a large multisubunit motor protein that moves various cargoes toward the minus ends of microtubules. In addition to the previously identified heavy, intermediate, and light intermediate chains, it has recently been recognized that cytoplasmic dynein also has several light chain subunits with apparent molecular weights between 8-20 kDa. To systematically identify the light chains of purified rat brain cytoplasmic dynein, peptide sequences were obtained from each light chain band resolved by gel electrophoresis. Both members of the tctex1 light chain family, tctex1 and rp3, were identified in a single band. Only one member of the roadblock family, roadblock-2, was found. Two members of the LC8 family were resolved as separate bands, the previously identified LC8 subunit, and a second novel cytoplasmic dynein family member, LC8b. The tissue distribution of these two dynein LC8 subunits differed, although LC8b was the major family member in brain. Database searches found that both LC8a and LC8b were also present in several mammalian species, and a third mammalian LC8 sequence, LC8c was found in the human database. The amino acid sequences of both LC8a and LC8b were completely conserved in mammals. LC8a and LC8b differ in only six of the 89 amino acids. The amino acid differences between LC8a and LC8b were located near the N-terminus of the molecules, and most were in the outward facing alpha-helices of the LC8 dimer. When the mammalian LC8a sequence was compared to the LC8 sequences found in six other animal species including Xenopus and Drosophila, there was, on average, 94% sequence identity. More variation was found in LC8 sequences obtained from plants, fungi, and parasites. LC8c differed from the other two human LC8 sequences in that it has amino acid substitutions in the intermediate chain binding domain at the C-terminal of the molecule. The position of amino acid substitutions of the three mammalian LC8 family members is consistent with the hypothesis that they bind to different proteins.

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Section: Methodsmentioning

confidence: 99%

Light chains of mammalian cytoplasmic dynein: identification and characterization of a family of LC8 light chains

Wilson

Salata

Susalka

et al. 2001

Cell Motil. Cytoskeleton

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“…The ubiquitous IC2C isoform of dynein IC was used in all work (Vaughan and Vallee, 1995;Brill and Pfister, 2000). Full-length mouse IC2C (IC2-FL) was cloned by polymerase chain reaction (PCR) with primers that added BglII and HindIII sites onto the ends of the IC2C expressed sequence tag (EST AA667372; Image clone no.…”

Section: Expression Constructsmentioning

confidence: 99%

Analysis of the Dynein-Dynactin Interaction In Vitro and In Vivo

King

Brown

Maier

et al. 2003

MBoC

164

196

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Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms. INTRODUCTIONCytoplasmic dynein is a minus end-directed, microtubulebased motor that provides the force for translocation, tension, and organization of cellular components. Each cytoplasmic dynein molecule contains two enzymatically active heads that convert the energy of ATP hydrolysis into mechanical work. These are connected to a basal cargo binding unit (for reviews of dynein structure, see King, 2000a,b). Cytoplasmic dynein requires another multisubunit protein complex, dynactin, for full activity (Gill et al., 1991;Schroer and Sheetz, 1991;Boylan et al., 2000). Dynactin contributes to cytoplasmic dynein function by acting as an "adapter" that expands the range of cargoes dynein can move (for review, see Karki and Holzbaur, 1999) and by increasing motor processivity (King and Schroer, 2000). Dynactin can also act independently of cytoplasmic dynein to anchor microtubules at the centrosome (Quintyne and Schroer, 2002). Dynactin is thought to accomplish these diverse tasks by using its distinct cargo-, cytoplasmic dynein-and microtubulebinding domains.The interaction between dynein and dynactin seems to be tightly regulated, because dynactin and dynein are not always colocalized in cells (for reviews, see Holleran et al., 1998;Karki and Holzbaur, 1999). For example, dynactin is concentrated at centrosomes throughout the cell cycle, whereas dynein accumulates between the time of centriole duplication (mid-S phase) and the onset of mitosis (Quintyne and Schroer, 2002). Both proteins are found at kinetochores, spindle poles, and along spindle microtubules during mitosis, but after mitosis dynein is lost from the centrosome, whereas dynactin remains. Similarly, dynactin, but not dynein, associates with microtubule plus ends (Vaughan et al., 1999;Habermann et al., 2001). Finally, dynein and dynactin colocalize on numerous endomembranes destined to move along microtubules (Habermann et al., 2001) and decrease in concert at the onset of mitosis (Niclas et al., 1996). Apparently, dynein and dynactin can be either associated or separate in cells, which highlights the importance of understanding how dynein...

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“…Biochemical hegeterogeneity also exists for the multisubunit motor protein CDyn [reviewed in (Brill and Pfister, 2000; Vallee et al, 2004)] (Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

“…1). In mammals, the main core of this multisubunit motor protein complex features two dynein heavy chain (DHCs), and two intermediate chain (DIC) subunits (Brill and Pfister, 2000). DHCs are responsible for the ATPase and microtuble-binding activities of CDyn, whereas DICs play a role on the binding of CDyn to a wide variety of MBO cargoes (Brill and Pfister, 2000; Vallee et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

“…In mammals, the main core of this multisubunit motor protein complex features two dynein heavy chain (DHCs), and two intermediate chain (DIC) subunits (Brill and Pfister, 2000). DHCs are responsible for the ATPase and microtuble-binding activities of CDyn, whereas DICs play a role on the binding of CDyn to a wide variety of MBO cargoes (Brill and Pfister, 2000; Vallee et al, 2004). In addition, CDyn can include various other subunits, including light intermediate chains (LICs) and various light chain subnits including LC8, roadblock and T-complex testis-specific protein 1 (TCTEX1) (Vallee et al, 2004), although the specific stoichiometries are unclear.…”

Section: Introductionmentioning

confidence: 99%

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Regulation of motor proteins, axonal transport deficits and adult-onset neurodegenerative diseases

Brady

Morfini

2017

Neurobiology of Disease

117

123

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Neurons affected in a wide variety of unrelated adult-onset neurodegenerative diseases (AONDs) typically exhibit a “dying back” pattern of degeneration, which is characterized by early deficits in synaptic function and neuritic pathology long before neuronal cell death. Consistent with this observation, multiple unrelated AONDs including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and several motor neuron diseases feature early alterations in kinase-based signaling pathways associated with deficits in axonal transport (AT), a complex cellular process involving multiple intracellular trafficking events powered by microtubule-based motor proteins. These pathogenic events have important therapeutic implications, suggesting that a focus on preservation of neuronal connections may be more effective to treat AONDs than addressing neuronal cell death. While the molecular mechanisms underlying AT abnormalities in AONDs are still being analyzed, evidence has accumulated linking those to a well-established pathological hallmark of multiple AONDs: altered patterns of neuronal protein phosphorylation. Here, we present a short overview on the biochemical heterogeneity of major motor proteins for AT, their regulation by protein kinases, and evidence revealing cell type-specific AT specializations. When considered together, these findings may help explain how independent pathogenic pathways can affect AT differentially in the context of each AOND.

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Biochemical and Molecular Analysis of the Mammalian Cytoplasmic Dynein Intermediate Chain

Cited by 15 publications

References 45 publications

Light chains of mammalian cytoplasmic dynein: identification and characterization of a family of LC8 light chains

Light chains of mammalian cytoplasmic dynein: identification and characterization of a family of LC8 light chains

Analysis of the Dynein-Dynactin Interaction In Vitro and In Vivo

Regulation of motor proteins, axonal transport deficits and adult-onset neurodegenerative diseases

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