scholarly journals Specificity of Cytoplasmic Dynein Subunits in Discrete Membrane-trafficking Steps

2009 ◽  
Vol 20 (12) ◽  
pp. 2885-2899 ◽  
Author(s):  
Krysten J. Palmer ◽  
Helen Hughes ◽  
David J. Stephens
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Cytoplasmic Dynein ◽  
Dominant Negative ◽  
Automated Image Analysis ◽  
Recycling Endosome ◽  
Microtubule Network ◽  
Dynein Motor ◽  
Specific Subset ◽  
Biochemical Analyses

The cytoplasmic dynein motor complex is known to exist in multiple forms, but few specific functions have been assigned to individual subunits. A key limitation in the analysis of dynein in intact mammalian cells has been the reliance on gross perturbation of dynein function, e.g., inhibitory antibodies, depolymerization of the entire microtubule network, or the use of expression of dominant negative proteins that inhibit dynein indirectly. Here, we have used RNAi and automated image analysis to define roles for dynein subunits in distinct membrane-trafficking processes. Depletion of a specific subset of dynein subunits, notably LIC1 (DYNC1LI1) but not LIC2 (DYNC1LI2), recapitulates a direct block of ER export, revealing that dynein is required to maintain the steady-state composition of the Golgi, through ongoing ER-to-Golgi transport. Suppression of LIC2 but not of LIC1 results in a defect in recycling endosome distribution and cytokinesis. Biochemical analyses also define the role of each subunit in stabilization of the dynein complex; notably, suppression of DHC1 or IC2 results in concomitant loss of Tctex1. Our data demonstrate that LIC1 and LIC2 define distinct dynein complexes that function at the Golgi versus recycling endosomes, respectively, suggesting that functional populations of dynein mediate discrete intracellular trafficking pathways.

scholarly journals New Perspectives on SNARE Function in the Yeast Minimal Endomembrane System

Genes ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 899 ◽  
Author(s):  
James H. Grissom ◽  
Verónica A. Segarra ◽  
Richard J. Chi
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Budding Yeast ◽  
Endocytic Pathway ◽  
Model Organisms ◽  
Endosomal Trafficking ◽  
Endomembrane System ◽  
Recycling Endosome ◽  
New Findings

Saccharomyces cerevisiae is one of the best model organisms for the study of endocytic membrane trafficking. While studies in mammalian cells have characterized the temporal and morphological features of the endocytic pathway, studies in budding yeast have led the way in the analysis of the endosomal trafficking machinery components and their functions. Eukaryotic endomembrane systems were thought to be highly conserved from yeast to mammals, with the fusion of plasma membrane-derived vesicles to the early or recycling endosome being a common feature. Upon endosome maturation, cargos are then sorted for reuse or degraded via the endo-lysosomal (endo-vacuolar in yeast) pathway. However, recent studies have shown that budding yeast has a minimal endomembrane system that is fundamentally different from that of mammalian cells, with plasma membrane-derived vesicles fusing directly to a trans-Golgi compartment which acts as an early endosome. Thus, the Golgi, rather than the endosome, acts as the primary acceptor of endocytic vesicles, sorting cargo to pre-vacuolar endosomes for degradation. The field must now integrate these new findings into a broader understanding of the endomembrane system across eukaryotes. This article synthesizes what we know about the machinery mediating endocytic membrane fusion with this new model for yeast endomembrane function.

scholarly journals Cooperative Accumulation of Dynein-Dynactin at Microtubule Minus-Ends Drives Microtubule Network Reorganization

2017 ◽  
Author(s):  
Ruensern Tan ◽  
Peter J. Foster ◽  
Daniel J. Needleman ◽  
Richard J. McKenney
Keyword(s):  
Mitotic Spindle ◽  
Large Scale ◽  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
List Type ◽  
Microtubule Network ◽  
Cellular Processes ◽  
Dynein Motor ◽  
Mitotic Spindle Assembly ◽  
Orientation Bias

SummaryCytoplasmic dynein-1 (dynein) is minus-end directed motor protein that transports cargo over long distances and organizes microtubules (MTs) during critical cellular processes such as mitotic spindle assembly. How dynein motor activity is harnessed for these diverse functions remains unknown. Here, we have uncovered a mechanism for how processive dynein-dynactin complexes drive MT-MT sliding, reorganization, and focusing, activities required for mitotic spindle assembly. We find that motors cooperatively accumulate, in limited numbers, at MT minus-ends. Minus-end accumulations drive MT-MT sliding, independent of MT orientation, and this activity always results in the clustering of MT minus-ends. At a mesoscale level, activated dynein-dynactin drives the formation and coalescence of MT asters. Macroscopically, dynein-dynactin activity leads to bulk contraction of millimeter-scale MT networks, demonstrating that minus-end accumulations produce network scale contractile stresses. Our data provides a model for how localized dynein activity is harnessed by cells to produce contractile stresses within the mitotic spindle.HighlightsProcessive dynein-dynactin complexes cooperatively form stable accumulations at MT minus-ends.Minus-end accumulations of motors slide MTs without orientation bias, leading to minus-end focusing.Minus-end accumulations of motors organize dynamic MTs into asters.Minus-end accumulations of motors drive bulk contractions of large-scale MT networks.

ARF6 requirement for Rac ruffling suggests a role for membrane trafficking in cortical actin rearrangements

1999 ◽  
Vol 112 (6) ◽  
pp. 855-866 ◽  
Author(s):  
H. Radhakrishna ◽  
O. Al-Awar ◽  
Z. Khachikian ◽  
J.G. Donaldson
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Dominant Negative ◽  
Aluminum Fluoride ◽  
Cortical Actin ◽  
Primary Human Fibroblast ◽  
Recycling Endosome ◽  
Gtp Binding ◽  
Rho Family ◽  
In Cells

The ARF6 GTPase regulates a novel endosomal-plasma membrane recycling pathway and influences cortical actin remodeling. Here we examined the relationship between ARF6 and Rac1, a Rho family GTPase, implicated in cortical actin rearrangements. Endogenous Rac1 colocalized with ARF6 at the plasma membrane and on the ARF6 recycling endosome in untransfected HeLa and primary human fibroblast cells. In transfected HeLa cells Rac1 and ARF6 also colocalized. Cells expressing wild-type ARF6 or Rac1 formed actin-containing surface protrusions and membrane ruffles, respectively, upon treatment with the G protein activator aluminum fluoride. Aluminum fluoride-treatment of cells transfected with equivalent amounts of plasmid resulted in enhanced membrane ruffling, with protrusions appearing as Rac expression was lowered. Co-expression of the dominant negative, GTP binding-defective ARF6 T27N mutant inhibited the aluminum fluoride-induced ruffling observed in cells expressing Rac1, and the constitutive ruffling observed in cells expressing the activated Rac1 Q61L mutant. In contrast, co-expression of the GTP-binding-defective, T17N mutant of either Rac1 or Cdc42 with ARF6 did not inhibit the aluminum fluoride-induced surface protrusions, nor did inactivation of Rho with C3-transferase. These observations suggest that ARF6, a non-Rho family GTPase, can, by itself, alter cortical actin and can influence the ability of Rac1 to form lamellipodia, in part, by regulating its trafficking to the plasma membrane.

scholarly journals mTORC1 activates SREBP-2 by suppressing cholesterol trafficking to lysosomes in mammalian cells

2017 ◽  
Vol 114 (30) ◽  
pp. 7999-8004 ◽  
Author(s):  
Walaa Eid ◽  
Kristin Dauner ◽  
Kevin C. Courtney ◽  
AnneMarie Gagnon ◽  
Robin J. Parks ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Cholesterol Synthesis ◽  
Regulatory Element ◽  
Dominant Negative ◽  
Cholesterol Trafficking ◽  
Endosomal Recycling ◽  
Sterol Regulatory Element ◽  
Dual Blockade ◽  
Mtorc1 Activation

mTORC1 is known to activate sterol regulatory element-binding proteins (SREBPs) including SREBP-2, a master regulator of cholesterol synthesis. Through incompletely understood mechanisms, activated mTORC1 triggers translocation of SREBP-2, an endoplasmic reticulum (ER) resident protein, to the Golgi where SREBP-2 is cleaved to translocate to the nucleus and activate gene expression for cholesterol synthesis. Low ER cholesterol is a well-established trigger for SREBP-2 activation. We thus investigated whether mTORC1 activates SREBP-2 by reducing cholesterol delivery to the ER. We report here that mTORC1 activation is accompanied by low ER cholesterol and an increase of SREBP-2 activation. Conversely, a decrease in mTORC1 activity coincides with a rise in ER cholesterol and a decrease in SERBP-2 activity. This rise in ER cholesterol is of lysosomal origin: blocking the exit of cholesterol from lysosomes by U18666A or NPC1 siRNA prevents ER cholesterol from increasing and, consequently, SREBP-2 is activated without mTORC1 activation. Furthermore, when mTORC1 activity is low, cholesterol is delivered to lysosomes through two membrane trafficking pathways: autophagy and rerouting of endosomes to lysosomes. Indeed, with dual blockade of both pathways by Atg5−/−and dominant-negative rab5, ER cholesterol fails to increase when mTORC1 activity is low, and SREBP-2 is activated. Conversely, overexpressing constitutively active Atg7, which forces autophagy and raises ER cholesterol even when mTORC1 activity is high, suppresses SREBP-2 activation. We conclude that mTORC1 actively suppresses autophagy and maintains endosomal recycling, thereby preventing endosomes and autophagosomes from reaching lysosomes. This results in a reduction of cholesterol in the ER and activation of SREBP-2.

scholarly journals Dynactin's pointed-end complex is a cargo-targeting module

2012 ◽  
Vol 23 (19) ◽  
pp. 3827-3837 ◽  
Author(s):  
Ting-Yu Yeh ◽  
Nicholas J. Quintyne ◽  
Brett R. Scipioni ◽  
D. Mark Eckley ◽  
Trina A. Schroer
Keyword(s):  
Cytoplasmic Dynein ◽  
Cross Linking ◽  
Structural Domain ◽  
Mitotic Spindles ◽  
Protein Overexpression ◽  
Recycling Endosome ◽  
Binding Partners ◽  
Dynein Motor ◽  
Pointed End ◽  
Chemical Cross Linking

Dynactin is an essential part of the cytoplasmic dynein motor that enhances motor processivity and serves as an adaptor that allows dynein to bind cargoes. Much is known about dynactin's interaction with dynein and microtubules, but how it associates with its diverse complement of subcellular binding partners remains mysterious. It has been suggested that cargo specification involves a group of subunits referred to as the “pointed-end complex.” We used chemical cross-linking, RNA interference, and protein overexpression to characterize interactions within the pointed-end complex and explore how it contributes to dynactin's interactions with endomembranes. The Arp11 subunit, which caps one end of dynactin's Arp1 filament, and p62, which binds Arp11 and Arp1, are necessary for dynactin stability. These subunits also allow dynactin to bind the nuclear envelope prior to mitosis. p27 and p25, by contrast, are peripheral components that can be removed without any obvious impact on dynactin integrity. Dynactin lacking these subunits shows reduced membrane binding. Depletion of p27 and p25 results in impaired early and recycling endosome movement, but late endosome movement is unaffected, and mitotic spindles appear normal. We conclude that the pointed-end complex is a bipartite structural domain that stabilizes dynactin and supports its binding to different subcellular structures.

scholarly journals Cytoplasmic dynein plays a role in mammalian mitotic spindle formation.

1993 ◽  
Vol 123 (4) ◽  
pp. 849-858 ◽  
Author(s):  
E A Vaisberg ◽  
M P Koonce ◽  
J R McIntosh
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Cytoplasmic Dynein ◽  
Detectable Effect ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Dynein Motor ◽  
Motor Enzymes ◽  
Chromosome Attachment

The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles, but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP-hydrolytic sites of the cytoplasmic dynein heavy chain from HeLa cells and from Dictyostelium. Monospecific antibodies have been raised to the resulting polypeptides, and these inhibit dynein motor activity in vitro. Their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells become arrested with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles that it may play are redundant. Possible mechanisms of dynein's involvement in mitosis are discussed.

Overexpression of normal and mutant Arp1alpha (centractin) differentially affects microtubule organization during mitosis and interphase

1999 ◽  
Vol 112 (20) ◽  
pp. 3507-3518 ◽  
Author(s):  
I.B. Clark ◽  
D.I. Meyer
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Cytoplasmic Dynein ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
Mitotic Apparatus ◽  
Mutant Proteins ◽  
Cell Extracts ◽  
Dynactin Complex

Dynactin is a large multisubunit complex that regulates cytoplasmic dynein-mediated functions. To gain insight into the role of dynactin's most abundant component, Arp1alpha was transiently overexpressed in mammalian cells. Arp1alpha overexpression resulted in a cell cycle delay at prometaphase. Intracellular dynactin, dynein and nuclear/mitotic apparatus (NuMA) protein were recruited to multiple foci associated with ectopic cytoplasmic aggregates of Arp1alpha in transfected cells. These ectopic aggregates nucleated supernumerary microtubule asters at prometaphase. Point mutations were generated in Arp1alpha that identified specific amino acids required for the prometaphase delay and for the formation of supernumerary microtubule asters. The mutant Arp1alpha proteins formed aggregates in cells that colocalized with dynactin and dynein peptides, but in contrast to wild-type Arp1alpha, NuMA localization remained unaffected. Although expression of mutant Arp1alpha proteins had no effect on mitotic cells, in interphase cells expression of the mutants resulted in disruption of the microtubule network. Immunoprecipitation studies demonstrated that overexpressed Arp1alpha interacts with dynactin and NuMA proteins in cell extracts, and that these interactions are destabilized in the Arp1alpha mutants. We conclude that the amino acids altered in the Arp1alpha mutant proteins participate in stabilizing interactions between overexpressed Arp1alpha and components of the endogenous dynactin complex as well as the NuMA protein.

scholarly journals ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization

2002 ◽  
Vol 13 (9) ◽  
pp. 3078-3095 ◽  
Author(s):  
Annette L. Boman ◽  
Paul D. Salo ◽  
Melissa J. Hauglund ◽  
Nicole L. Strand ◽  
Shelly J. Rensink ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Carboxypeptidase Y ◽  
Minor Role ◽  
Temperature Sensitive ◽  
Adp Ribosylation ◽  
And Function ◽  
Adp Ribosylation Factor

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

Sterol and lipid trafficking in mammalian cells

2006 ◽  
Vol 34 (3) ◽  
pp. 335-339 ◽  
Author(s):  
F.R. Maxfield ◽  
M. Mondal
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Vesicular Transport ◽  
Cholesterol Content ◽  
Cellular Functions ◽  
Lipid Trafficking ◽  
Sterol Transport ◽  
A Cell ◽  
Asymmetrically Distributed

The pathways involved in the intracellular transport and distribution of lipids in general, and sterols in particular, are poorly understood. Cholesterol plays a major role in modulating membrane bilayer structure and important cellular functions, including signal transduction and membrane trafficking. Both the overall cholesterol content of a cell, as well as its distribution in specific organellar membranes are stringently regulated. Several diseases, many of which are incurable at present, have been characterized as results of impaired cholesterol transport and/or storage in the cells. Despite their importance, many fundamental aspects of intracellular sterol transport and distribution are not well understood. For instance, the relative roles of vesicular and non-vesicular transport of cholesterol have not yet been fully determined, nor are the non-vesicular transport mechanisms well characterized. Similarly, whether cholesterol is asymmetrically distributed between the two leaflets of biological membranes, and if so, how this asymmetry is maintained, is poorly understood. In this review, we present a summary of the current understanding of these aspects of intracellular trafficking and distribution of lipids, and more specifically, of sterols.

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