scholarly journals RUFY3 links Arl8b and JIP4-Dynein complex to regulate lysosome size and positioning

2021 ◽  
Author(s):  
Gaurav Kumar ◽  
Prateek Chawla ◽  
Sanya Chadha ◽  
Sheetal Sharma ◽  
Kanupriya Sethi ◽  
...  
Keyword(s):  
G Protein ◽  
Spatial Organization ◽  
Adaptor Protein ◽  
Retrograde Transport ◽  
Anterograde Transport ◽  
Motor Complex ◽  
Gtp Binding ◽  
Microtubule Motor ◽  
Mobile Fraction ◽  
Dynactin Complex

Abstract The whole-cell scale spatial organization of lysosomes is regulated by their bidirectional motility on microtubule tracks. Small GTP-binding (G) protein, Arl8b, stimulates the anterograde transport of lysosomes by recruiting adaptor protein SKIP (also known as PLEKHM2), which in turn couples the microtubule motor kinesin-1. Here, we have identified an Arl8b effector, RUN and FYVE domain-containing protein family member 3, RUFY3, which drives the retrograde transport of lysosomes. Artificial targeting of RUFY3 to the surface of mitochondria was sufficient to drive their perinuclear positioning. We find that RUFY3 interacts with the JIP4-Dynein-Dynactin complex and mediates Arl8b association with the retrograde motor complex. The mobile fraction of the total lysosomes per cell was significantly enhanced upon RUFY3 depletion, suggesting that RUFY3 maintains the lysosomes clustering within the perinuclear cloud. Expectedly, RUFY3 knockdown disrupted the perinuclear positioning of lysosomes upon nutrient starvation and/or serum depletion, although lysosome continued to undergo fusion with autophagosomes. Interestingly, lysosome fission events were more frequent in RUFY3-depleted cells and accordingly, there was a striking reduction in lysosome size, an effect that was also observed in dynein and JIP4 depleted cells. These findings indicate that the dynein-dependent “perinuclear cloud” arrangement of lysosomes also regulates the size of these proteolytic compartments and, likely, their cellular roles.

scholarly journals Identification of an Axonal Kinesin-3 Motor for Fast Anterograde Vesicle Transport that Facilitates Retrograde Transport of Neuropeptides

2008 ◽  
Vol 19 (1) ◽  
pp. 274-283 ◽  
Author(s):  
Rosemarie V. Barkus ◽  
Olga Klyachko ◽  
Dai Horiuchi ◽  
Barry J. Dickson ◽  
William M. Saxton
Keyword(s):  
Transport Mechanism ◽  
Fluorescent Protein ◽  
Retrograde Transport ◽  
Major Contributor ◽  
Anterograde Transport ◽  
Microtubule Motor ◽  
Fast Transport ◽  
Green Fluorescent ◽  
Neuronal Functions ◽  
Axonal Swellings

A screen for genes required in Drosophila eye development identified an UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic anterograde axonal motor, kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of green fluorescent protein-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to anterograde transport of mitochondria, which are transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism.

scholarly journals Systematic dissection of dynein regulators in mitosis

2013 ◽  
Vol 201 (2) ◽  
pp. 201-215 ◽  
Author(s):  
Jonne A. Raaijmakers ◽  
Marvin E. Tanenbaum ◽  
René H. Medema
Keyword(s):  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Cytoplasmic Dynein ◽  
Comprehensive Overview ◽  
Motor Complex ◽  
Essential Components ◽  
Complex Thought ◽  
Specific Activities ◽  
Dynactin Complex

Cytoplasmic dynein is a large minus end–directed motor complex with multiple functions during cell division. The dynein complex interacts with various adaptor proteins, including the dynactin complex, thought to be critical for most dynein functions. Specific activities have been linked to several subunits and adaptors, but the function of the majority of components has remained elusive. Here, we systematically address the function of each dynein–dynactin subunit and adaptor protein in mitosis. We identify the essential components that are required for all mitotic functions of dynein. Moreover, we find specific dynein recruitment factors, and adaptors, like Nde1/L1, required for activation, but largely dispensable for dynein localization. Most surprisingly, our data show that dynactin is not required for dynein-dependent spindle organization, but acts as a dynein recruitment factor. These results provide a comprehensive overview of the role of dynein subunits and adaptors in mitosis and reveal that dynein forms distinct complexes requiring specific recruiters and activators to promote orderly progression through mitosis.

scholarly journals Dynein/dynactin is necessary for anterograde transport of Mbp mRNA in oligodendrocytes and for myelination in vivo

2017 ◽  
Vol 114 (43) ◽  
pp. E9153-E9162 ◽  
Author(s):  
Amy L. Herbert ◽  
Meng-meng Fu ◽  
Catherine M. Drerup ◽  
Ryan S. Gray ◽  
Breanne L. Harty ◽  
...  
Keyword(s):  
Action Potentials ◽  
Animal Studies ◽  
Mrna Transport ◽  
Anterograde Transport ◽  
Protein Levels ◽  
Motor Complex ◽  
Mrna Granules ◽  
The Central Nervous System ◽  
Dynactin Complex

Oligodendrocytes in the central nervous system produce myelin, a lipid-rich, multilamellar sheath that surrounds axons and promotes the rapid propagation of action potentials. A critical component of myelin is myelin basic protein (MBP), expression of which requires anterograde mRNA transport followed by local translation at the developing myelin sheath. Although the anterograde motor kinesin KIF1B is involved in mbp mRNA transport in zebrafish, it is not entirely clear how mbp transport is regulated. From a forward genetic screen for myelination defects in zebrafish, we identified a mutation in actr10, which encodes the Arp11 subunit of dynactin, a critical activator of the retrograde motor dynein. Both the actr10 mutation and pharmacological dynein inhibition in zebrafish result in failure to properly distribute mbp mRNA in oligodendrocytes, indicating a paradoxical role for the retrograde dynein/dynactin complex in anterograde mbp mRNA transport. To address the molecular mechanism underlying this observation, we biochemically isolated reporter-tagged Mbp mRNA granules from primary cultured mammalian oligodendrocytes to show that they indeed associate with the retrograde motor complex. Next, we used live-cell imaging to show that acute pharmacological dynein inhibition quickly arrests Mbp mRNA transport in both directions. Chronic pharmacological dynein inhibition also abrogates Mbp mRNA distribution and dramatically decreases MBP protein levels. Thus, these cell culture and whole animal studies demonstrate a role for the retrograde dynein/dynactin motor complex in anterograde mbp mRNA transport and myelination in vivo.

scholarly journals Dynactin Is Required for Coordinated Bidirectional Motility, but Not for Dynein Membrane Attachment

2007 ◽  
Vol 18 (6) ◽  
pp. 2081-2089 ◽  
Author(s):  
Marjan Haghnia ◽  
Valeria Cavalli ◽  
Sameer B. Shah ◽  
Kristina Schimmelpfeng ◽  
Richard Brusch ◽  
...  
Keyword(s):  
Molecular Motor ◽  
Retrograde Transport ◽  
Anterograde Transport ◽  
Membrane Attachment ◽  
Membrane Compartments ◽  
Human Motor ◽  
Neuronal Vesicles ◽  
Dynactin Complex ◽  
Lateral Sclerosis

Transport of cellular and neuronal vesicles, organelles, and other particles along microtubules requires the molecular motor protein dynein ( Mallik and Gross, 2004 ). Critical to dynein function is dynactin, a multiprotein complex commonly thought to be required for dynein attachment to membrane compartments ( Karki and Holzbaur, 1999 ). Recent work also has found that mutations in dynactin can cause the human motor neuron disease amyotrophic lateral sclerosis ( Puls et al., 2003 ). Thus, it is essential to understand the in vivo function of dynactin. To test directly and rigorously the hypothesis that dynactin is required to attach dynein to membranes, we used both a Drosophila mutant and RNA interference to generate organisms and cells lacking the critical dynactin subunit, actin-related protein 1. Contrary to expectation, we found that apparently normal amounts of dynein associate with membrane compartments in the absence of a fully assembled dynactin complex. In addition, anterograde and retrograde organelle movement in dynactin deficient axons was completely disrupted, resulting in substantial changes in vesicle kinematic properties. Although effects on retrograde transport are predicted by the proposed function of dynactin as a regulator of dynein processivity, the additional effects we observed on anterograde transport also suggest potential roles for dynactin in mediating kinesin-driven transport and in coordinating the activity of opposing motors ( King and Schroer, 2000 ).

scholarly journals The small GTP-binding protein rab6 functions in intra-Golgi transport.

1994 ◽  
Vol 127 (6) ◽  
pp. 1575-1588 ◽  
Author(s):  
O Martinez ◽  
A Schmidt ◽  
J Salaméro ◽  
B Hoflack ◽  
M Roa ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Transport ◽  
Binding Protein ◽  
Retrograde Transport ◽  
Wild Type ◽  
Gtp Binding Protein ◽  
Anterograde Transport ◽  
Golgi Transport ◽  
Gtp Binding ◽  
Hemagglutinin Protein

Rab6 is a ubiquitous ras-like GTP-binding protein associated with the membranes of the Golgi complex (Goud, B., A. Zahraoui, A. Tavitian, and J. Saraste. 1990. Nature (Lond.). 345:553-556; Antony, C., C. Cibert, G. Géraud, A. Santa Maria, B. Maro, V. Mayau, and B. Goud. 1992. J. Cell Sci. 103: 785-796). We have transiently overexpressed in mouse L cells and human HeLa cells wild-type rab6, GTP (rab6 Q72L), and GDP (rab6 T27N) -bound mutants of rab6 and analyzed the intracellular transport of a soluble secreted form of alkaline phosphatase (SEAP) and of a plasma membrane protein, the hemagglutinin protein (HA) of influenza virus. Over-expression of wild-type rab6 and rab6 Q72L greatly reduced transport of both markers between cis/medial (alpha-mannosidase II positive) and late (sialyl-transferase positive) Golgi compartments, without affecting transport from the endoplasmic reticulum (ER) to cis/medial-Golgi or from the trans-Golgi network (TGN) to the plasma membrane. Whereas overexpression of rab6 T27N did not affect the individual steps of transport between ER and the plasma membrane, it caused an apparent delay in secretion, most likely due to the accumulation of the transport markers in late Golgi compartments. Overexpression of both rab6 Q72L and rab6 T27N altered the morphology of the Golgi apparatus as well as that of the TGN, as assessed at the immunofluorescence level with several markers. We interpret these results as indicating that rab6 controls intra-Golgi transport, either acting as an inhibitor in anterograde transport or as a positive regulator of retrograde transport.

Disturbing Gtp-Binding Protein Function Through Microinjection Into The Visual Cell Of Limulus

1992 ◽  
Vol 47 (11-12) ◽  
pp. 915-921 ◽  
Author(s):  
Henmg Stieve ◽  
Barbara Niemeyer ◽  
Klaus Aktories ◽  
Heidi E. Hamm
Keyword(s):  
Monoclonal Antibody ◽  
G Protein ◽  
Protein Function ◽  
Clostridium Botulinum ◽  
Functional Role ◽  
Electrical Response ◽  
Visual Cell ◽  
Gtp Binding ◽  
Dim Light ◽  
Ventral Nerve Photoreceptor

We have tested the action of three agents microinjected into the ventral nerve photoreceptor of Limulus on the electrical response to dim light. 1. A monoclonal antibody (mAb 4 A) against the Gɑ subunit of frog transducin reduces the size of the receptor current to 60%, suggesting an interaction with Gɑ in the Limulus photoreceptor. 2. Injection of Clostridium botulinum ADPribosyltransferase C 3 reduces the size to 46%; latency is not affected. The results imply that small GTP-binding proteins play a functional role in photoreception of invertebrates. 3. Injection of GD P-β-S reduces dose-dependently the size of the receptor current to 15% and prolongs the latency to 200%, presumably by reducing number and rate of G-protein activations

scholarly journals Vesicular Transport Is Not Required for the Cytoplasmic Pool of Cholera Toxin To Interact with the Stimulatory Alpha Subunit of the Heterotrimeric G Protein

2004 ◽  
Vol 72 (12) ◽  
pp. 6826-6835 ◽  
Author(s):  
Ken Teter ◽  
Michael G. Jobling ◽  
Randall K. Holmes
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cholera Toxin ◽  
G Protein ◽  
Vesicular Transport ◽  
Cytotoxic Action ◽  
Anterograde Transport ◽  
Heterotrimeric G Protein ◽  
Α Subunit ◽  
Vesicular Traffic

ABSTRACT Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic A1 polypeptide of CT (CTA1) then crosses the ER membrane, enters the cytosol, ADP-ribosylates the stimulatory α subunit of the heterotrimeric G protein (Gsα) at the cytoplasmic face of the plasma membrane, and activates adenylate cyclase. The cytosolic pool of CTA1 may reach the plasma membrane and its Gsα target by traveling on anterograde-directed transport vesicles. We examined this possibility with the use of a plasmid-based transfection system that directed newly synthesized CTA1 to either the ER lumen or the cytosol of CHO cells. Such a system allowed us to bypass the CT retrograde trafficking itinerary from the cell surface to the ER. Previous work has shown that the ER-localized pool of CTA1 is rapidly exported from the ER to the cytosol. Expression of CTA1 in either the ER or the cytosol led to the activation of Gsα, and Gsα activation was not inhibited in transfected cells exposed to drugs that inhibit vesicular traffic. Thus, anterograde transport from the ER to the plasma membrane is not required for the cytotoxic action of CTA1.

scholarly journals Dynactin is required for bidirectional organelle transport

2003 ◽  
Vol 160 (3) ◽  
pp. 297-301 ◽  
Author(s):  
Sean W. Deacon ◽  
Anna S. Serpinskaya ◽  
Patricia S. Vaughan ◽  
Monica Lopez Fanarraga ◽  
Isabelle Vernos ◽  
...  
Keyword(s):  
Cell Types ◽  
Cytoplasmic Dynein ◽  
Opposite Polarity ◽  
Model System ◽  
Organelle Transport ◽  
Transport Activity ◽  
Microtubule Motors ◽  
Microtubule Motor ◽  
Dynactin Complex ◽  
Binding Subunit

Kinesin II is a heterotrimeric plus end–directed microtubule motor responsible for the anterograde movement of organelles in various cell types. Despite substantial literature concerning the types of organelles that kinesin II transports, the question of how this motor associates with cargo organelles remains unanswered. To address this question, we have used Xenopus laevis melanophores as a model system. Through analysis of kinesin II–mediated melanosome motility, we have determined that the dynactin complex, known as an anchor for cytoplasmic dynein, also links kinesin II to organelles. Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II–associated protein (XKAP), binds directly to the p150Glued subunit of dynactin. This interaction occurs through aa 530–793 of XKAP and aa 600–811 of p150Glued. These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.

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