scholarly journals Dynein, Dynactin, and Kinesin II's Interaction with Microtubules Is Regulated during Bidirectional Organelle Transport

2000 ◽  
Vol 151 (1) ◽  
pp. 155-166 ◽  
Author(s):  
Eric L. Reese ◽  
Leah T. Haimo
Keyword(s):  
Pigment Granule ◽  
Cytoplasmic Dynein ◽  
Binding Activity ◽  
The State ◽  
Organelle Transport ◽  
Microtubule Binding ◽  
Pigment Granules ◽  
Microtubule Motors

The microtubule motors, cytoplasmic dynein and kinesin II, drive pigmented organelles in opposite directions in Xenopus melanophores, but the mechanism by which these or other motors are regulated to control the direction of organelle transport has not been previously elucidated. We find that cytoplasmic dynein, dynactin, and kinesin II remain on pigment granules during aggregation and dispersion in melanophores, indicating that control of direction is not mediated by a cyclic association of motors with these organelles. However, the ability of dynein, dynactin, and kinesin II to bind to microtubules varies as a function of the state of aggregation or dispersion of the pigment in the cells from which these molecules are isolated. Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment. Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo. These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.

scholarly journals Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

2018 ◽  
Vol 315 (5) ◽  
pp. E924-E948 ◽  
Author(s):  
Qing Wen ◽  
Elizabeth I. Tang ◽  
Wing-yee Lui ◽  
Will M. Lee ◽  
Chris K. C. Wong ◽  
...  
Keyword(s):  
Germ Cell ◽  
Heavy Chain ◽  
Sertoli Cells ◽  
Cytoplasmic Dynein ◽  
Seminiferous Epithelium ◽  
Organelle Transport ◽  
Cellular Events

In the mammalian testis, spermatogenesis is dependent on the microtubule (MT)-specific motor proteins, such as dynein 1, that serve as the engine to support germ cell and organelle transport across the seminiferous epithelium at different stages of the epithelial cycle. Yet the underlying molecular mechanism(s) that support this series of cellular events remain unknown. Herein, we used RNAi to knockdown cytoplasmic dynein 1 heavy chain (Dync1h1) and an inhibitor ciliobrevin D to inactivate dynein in Sertoli cells in vitro and the testis in vivo, thereby probing the role of dynein 1 in spermatogenesis. Both treatments were shown to extensively induce disruption of MT organization across Sertoli cells in vitro and the testis in vivo. These changes also perturbed the transport of spermatids and other organelles (such as phagosomes) across the epithelium. These changes thus led to disruption of spermatogenesis. Interestingly, the knockdown of dynein 1 or its inactivation by ciliobrevin D also perturbed gross disruption of F-actin across the Sertoli cells in vitro and the seminiferous epithelium in vivo, illustrating there are cross talks between the two cytoskeletons in the testis. In summary, these findings confirm the role of cytoplasmic dynein 1 to support the transport of spermatids and organelles across the seminiferous epithelium during the epithelial cycle of spermatogenesis.

scholarly journals Identification of a novel nucleotide-sensitive microtubule-binding protein in HeLa cells.

1990 ◽  
Vol 110 (5) ◽  
pp. 1623-1633 ◽  
Author(s):  
J E Rickard ◽  
T E Kreis
Keyword(s):  
Hela Cells ◽  
High Speed ◽  
Binding Proteins ◽  
Motor Proteins ◽  
Cytoplasmic Dynein ◽  
Microtubule Binding ◽  
Linear Arrays ◽  
Immunofluorescence Labeling

A protein of Mr 170,000 (170K protein) has been identified in HeLa cells, using an antiserum raised against HeLa nucleotide-sensitive microtubule-binding proteins. Affinity-purified antibodies specific for this 170K polypeptide were used for its characterization. In vitro sedimentation of the 170K protein with taxol microtubules polymerized from HeLa high-speed supernatant is enhanced in the presence of an ATP depleting system, but unaffected by the non-hydrolyzable ATP analogue AMP-PNP. In addition, it can be eluted from taxol microtubules by ATP or GTP, as well as NaCl. Thus it shows microtubule-binding characteristics distinct from those of the previously described classes of nucleotide-sensitive microtubule-binding proteins, the motor proteins kinesin and cytoplasmic dynein, homologues of which are also present in HeLa cells. The 170K protein sediments on sucrose gradients at approximately 6S, separate from kinesin (9.5S) and cytoplasmic dynein (20S), further indicating that it is not associated with these motor proteins. Immunofluorescence localization of the 170K protein shows a patchy distribution in interphase HeLa cells, often organized into linear arrays that correlate with microtubules. However, not all microtubules are labeled, and there is a significant accumulation of antigen at the peripheral ends of microtubules. In mitotic cells, 170K labeling is found in the spindle, but there is also dotty labeling in the cytoplasm. After depolymerization of microtubules by nocodazole, the staining pattern is also patchy but not organized in linear arrays, suggesting that the protein may be able to associate with other intracellular structures as well as microtubules. In vinblastine-treated cells, there is strong labeling of tubulin paracrystals, and random microtubules induced in vivo by taxol are also labeled by the antibodies. These immunofluorescence labeling patterns are stable to extraction of cells with Triton X-100 before fixation, further suggesting an association of the protein with cytoplasmic structures. In vivo, therefore, the 170K protein appears to be associated with a subset of microtubules at discrete sites. Its in vitro behavior suggests that it belongs to a novel class of nucleotide-sensitive microtubule-binding proteins.

scholarly journals Lis1 has two opposing modes of regulating cytoplasmic dynein

2017 ◽  
Author(s):  
Morgan E. DeSantis ◽  
Michael A. Cianfrocco ◽  
Zaw Min Htet ◽  
Phuoc Tien Tran ◽  
Samara L. Reck-Peterson ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intracellular Transport ◽  
Cytoplasmic Dynein ◽  
The Novel ◽  
Microtubule Binding ◽  
In Vivo Experiments ◽  
Cryo Electron Microscopy ◽  
Functional Versatility

SummaryRegulation is central to the functional versatility of cytoplasmic dynein, a motor involved in intracellular transport, cell division, and neurodevelopment. Previous work established that Lis1, a conserved and ubiquitous regulator of dynein, binds to its motor domain and induces a tight microtubule-binding state in dynein. The work we present here—a combination of biochemistry, single-molecule assays, cryo-electron microscopy and in vivo experiments—led to the surprising discovery that Lis1 has two opposing modes of regulating dynein, being capable of inducing both low and high affinity for the microtubule. We show that these opposing modes depend on the stoichiometry of Lis1 binding to dynein and that this stoichiometry is regulated by the nucleotide state of dynein’s AAA3 domain. We present data on the in vitro and in vivo consequences of abolishing the novel Lis1-induced weak microtubule-binding state in dynein and propose a new model for the regulation of dynein by Lis1.

scholarly journals Centromere function on minichromosomes isolated from budding yeast.

1993 ◽  
Vol 4 (8) ◽  
pp. 859-870 ◽  
Author(s):  
J Kingsbury ◽  
D Koshland
Keyword(s):  
Budding Yeast ◽  
Binding Activity ◽  
Chromosomal Dna ◽  
Microtubule Binding ◽  
Specific Factors ◽  
Stable Core ◽  
Insight Into ◽  
Binding Component

Centromeres are a complex of centromere DNA (CEN DNA) and specific factors that help mediate microtubule-dependent movement of chromosomes during mitosis. Minichromosomes can be isolated from budding yeast in a way that their centromeres retain the ability to bind microtubules in vitro. Here, we use the binding of these minichromosomes to microtubules to gain insight into the properties of centromeres assembled in vivo. Our results suggest that neither chromosomal DNA topology nor proximity of telomeres influence the cell's ability to assemble centromeres with microtubule-binding activity. The microtubule-binding activity of the minichromosome's centromere is stable in the presence of competitor CEN DNA, suggesting that the complex between the minichromosome CEN DNA and proteins directly bound to it is very stable. The efficiency of centromere binding to microtubules is dependent upon the concentration of microtubule polymer and is inhibited by ATP. These properties are similar to those exhibited by mechanochemical motors. The binding of minichromosomes to microtubules can be inactivated by the presence of 0.2 M NaCl and then reactivated by restoring NaCl to 0.1 M. In 0.2 M NaCl, some centromere factor(s) bind to microtubules, whereas other(s) apparently remain bound to the minichromosome's CEN DNA. Therefore, the yeast centromere appears to consist of two domains: the first consists of a stable core containing CEN DNA and CEN DNA-binding proteins; the second contains a microtubule-binding component(s). The molecular functions of this second domain are discussed.

scholarly journals A role for regulated binding of p150Glued to microtubule plus ends in organelle transport

2002 ◽  
Vol 158 (2) ◽  
pp. 305-319 ◽  
Author(s):  
Patricia S. Vaughan ◽  
Pedro Miura ◽  
Matthew Henderson ◽  
Belinda Byrne ◽  
Kevin T. Vaughan
Keyword(s):  
Phosphorylation Site ◽  
Binding Specificity ◽  
Time Lapse ◽  
Cytoplasmic Dynein ◽  
Live Cell ◽  
Organelle Transport ◽  
Dynamic Binding ◽  
Golgi Membranes

A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed p150Glued by live-cell imaging. Time-lapse analysis of p150Glued revealed targeting to the plus ends of growing microtubules, requiring the NH2-terminal cytoskeleton-associated protein–glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested p150Glued phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of p150Glued phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that p150Glued phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-p150Glued during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-p150Glued–labeled microtubules just prior to transport, implicating microtubules and dynactin in a search–capture mechanism for minus-end–directed organelles.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

Microtubule motors and other maps

1990 ◽  
Vol 48 (3) ◽  
pp. 1-1
Author(s):  
Richard B. Vallee
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Structural Components ◽  
Microtubule Associated Proteins ◽  
Microtubule Motors ◽  
Associated Proteins ◽  
The Subject ◽  
Intracellular Motility

Microtubules are involved in a number of forms of intracellular motility, including mitosis and bidirectional organelle transport. Purified microtubules from brain and other sources contain tubulin and a diversity of microtubule associated proteins (MAPs). Some of the high molecular weight MAPs - MAP 1A, 1B, 2A, and 2B - are long, fibrous molecules that serve as structural components of the cytamatrix. Three MAPs have recently been identified that show microtubule activated ATPase activity and produce force in association with microtubules. These proteins - kinesin, cytoplasmic dynein, and dynamin - are referred to as cytoplasmic motors. The latter two will be the subject of this talk.Cytoplasmic dynein was first identified as one of the high molecular weight brain MAPs, MAP 1C. It was determined to be structurally equivalent to ciliary and flagellar dynein, and to produce force toward the minus ends of microtubules, opposite to kinesin.

scholarly journals C-Terminal Deletions Can Suppress Temperature-Sensitive Mutations and Change Dominance in the Phage Mu Repressor

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 661-672 ◽  
Author(s):  
Jodi L Vogel ◽  
Vincent Geuskens ◽  
Lucie Desmet ◽  
N Patrick Higgins ◽  
Ariane Toussaint
Keyword(s):  
Binding Activity ◽  
Temperature Sensitive ◽  
Dna Binding Activity ◽  
Heat Stable ◽  
C Terminus ◽  
Acid Domain ◽  
Mutant Forms ◽  
Amino Acid Domain

Abstract Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup° hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup° hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179–V196), binds Mu operator DNA more stably at 42° in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.

Tocotrienols and Cancer: From the State of the Art to Promising Novel Patents

2019 ◽  
Vol 14 (1) ◽  
pp. 5-18 ◽  
Author(s):  
Fabrizio Fontana ◽  
Michela Raimondi ◽  
Monica Marzagalli ◽  
Roberta M. Moretti ◽  
Marina Montagnani Marelli ◽  
...  
Keyword(s):  
Cancer Prevention ◽  
State Of The Art ◽  
Synergistic Effects ◽  
The State ◽  
Therapeutic Interventions ◽  
Multiple Tumor ◽  
Anti Cancer ◽  
Important Health

Background: Tocotrienols (TTs) are vitamin E derivatives naturally occurring in several plants and vegetable oils. Like Tocopherols (TPs), they comprise four isoforms, α, β, γ and δ, but unlike TPs, they present an unsaturated isoprenoid chain. Recent studies indicate that TTs provide important health benefits, including neuroprotective, anti-inflammatory, anti-oxidant, cholesterol lowering and immunomodulatory effects. Moreover, they have been found to possess unique anti-cancer properties.Objective:The purpose of this review is to present an overview of the state of the art of TTs role in cancer prevention and treatment, as well as to describe recent patents proposing new methods for TTs isolation, chemical modification and use in cancer prevention and/or therapy.Methods:Recent literature and patents focusing on TTs anti-cancer applications have been identified and reviewed, with special regard to their scientific impact and novelty.Results:TTs have demonstrated significant anti-cancer activity in multiple tumor types, both in vitro and in vivo. Furthermore, they have shown synergistic effects when given in combination with standard anti-cancer agents or other anti-tumor natural compounds. Finally, new purification processes and transgenic sources have been designed in order to improve TTs production, and novel TTs formulations and synthetic derivatives have been developed to enhance their solubility and bioavailability.Conclusion:The promising anti-cancer effects shown by TTs in several preclinical studies may open new opportunities for therapeutic interventions in different tumors. Thus, clinical trials aimed at confirming TTs chemopreventive and tumor-suppressing activity, particularly in combination with standard therapies, are urgently needed.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1056-1067 ◽  
Author(s):  
Mira T. Kassouf ◽  
Hedia Chagraoui ◽  
Paresh Vyas ◽  
Catherine Porcher
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Experimental Systems ◽  
Dna Binding Activity ◽  
Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

Sign in / Sign up

Export Citation Format

Share Document