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 Intraflagellar Transport and Functional Analysis of Genes Required for Flagellum Formation in Trypanosomes

2008 ◽  
Vol 19 (3) ◽  
pp. 929-944 ◽  
Author(s):  
Sabrina Absalon ◽  
Thierry Blisnick ◽  
Linda Kohl ◽  
Géraldine Toutirais ◽  
Gwénola Doré ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Anterograde Transport ◽  
Body Regions ◽  
Cilia And Flagella ◽  
Bidirectional Movement ◽  
Green Fluorescent

Intraflagellar transport (IFT) is the bidirectional movement of protein complexes required for cilia and flagella formation. We investigated IFT by analyzing nine conventional IFT genes and five novel putative IFT genes (PIFT) in Trypanosoma brucei that maintain its existing flagellum while assembling a new flagellum. Immunostaining against IFT172 or expression of tagged IFT20 or green fluorescent protein GFP::IFT52 revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella. IFT particles were detected by electron microscopy and exhibited a strict localization to axonemal microtubules 3–4 and 7–8, suggesting the existence of specific IFT tracks. Rapid (>3 μm/s) bidirectional intraflagellar movement of GFP::IFT52 was observed in old and new flagella. RNA interference silencing demonstrated that all individual IFT and PIFT genes are essential for new flagellum construction but the old flagellum remained present. Inhibition of IFTB proteins completely blocked axoneme construction. Absence of IFTA proteins (IFT122 and IFT140) led to formation of short flagella filled with IFT172, indicative of defects in retrograde transport. Two PIFT proteins turned out to be required for retrograde transport and three for anterograde transport. Finally, flagellum membrane elongation continues despite the absence of axonemal microtubules in all IFT/PIFT mutant.

Lumenal targeted GFP, used as a marker of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular network

2000 ◽  
Vol 113 (18) ◽  
pp. 3151-3159 ◽  
Author(s):  
R. Blum ◽  
D.J. Stephens ◽  
I. Schulz
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Temperature Sensitive ◽  
Long Distance ◽  
Anterograde Transport ◽  
Network Transport ◽  
Vesicular Stomatitis ◽  
Green Fluorescent ◽  
Time Lapse Imaging ◽  
Tubular Membranes

The mechanism by which soluble proteins without sorting motifs are transported to the cell surface is not clear. Here we show that soluble green fluorescent protein (GFP) targeted to the lumen of the endoplasmic reticulum but lacking any known retrieval, retention or targeting motifs, was accumulated in the lumen of the ERGIC if cells were kept at reduced temperature. Upon activation of anterograde transport by rewarming of cells, lumenal GFP stained a microtubule-dependent, pre-Golgi tubulo-vesicular network that served as transport structure between peripheral ERGIC-elements and the perinuclear Golgi complex. Individual examples of these tubular elements up to 20 microm in length were observed. Time lapse imaging indicated rapid anterograde flow of soluble lumenal GFP through this network. Transport tubules, stained by lumenal GFP, segregated rapidly from COPI-positive membranes after transport activation. A transmembrane cargo marker, the temperature sensitive glycoprotein of the vesicular stomatitis virus, ts-045 G, is also not present in tubules which contained the soluble cargo marker lum-GFP. These results suggest a role for pre-Golgi vesicular tubular membranes in long distance anterograde transport of soluble cargo. http://www.biologists.com/JCS/movies/jcs1334.html

scholarly journals Retrograde Transport of Transcription Factor NF-κB in Living Neurons

2000 ◽  
Vol 276 (15) ◽  
pp. 11821-11829 ◽  
Author(s):  
Henning Wellmann ◽  
Barbara Kaltschmidt ◽  
Christian Kaltschmidt
Keyword(s):  
Transcription Factor ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Retrograde Transport ◽  
Receptor Activation ◽  
Transcriptional Changes ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Living Neurons

The mechanism by which signals such as those produced by glutamate are transferred to the nucleus may involve direct transport of an activated transcription factor to trigger long-term transcriptional changes. Ionotropic glutamate receptor activation or depolarization activates transcription factor NF-κB and leads to translocation of NF-κB from the cytoplasm to the nucleus. We investigated the dynamics of NF-κB translocation in living neurons by tracing the NF-κB subunit RelA (p65) with jellyfish green fluorescent protein. We found that green fluorescent protein-RelA was located in either the nucleus or cytoplasm and neurites, depending on the coexpression of the cognate inhibitor of NF-κB, IκB-α. Stimulation with glutamate, kainate, or potassium chloride resulted in a redistribution of NF-κB from neurites to the nucleus. This transport depended on an intact nuclear localization signal on RelA. Thus, in addition to its role as a transcription factor, NF-κB may be a signal transducer, transmitting transient glutamatergic signals from distant sites to the nucleus.

scholarly journals Pseudorabies Virus Us9 Directs Axonal Sorting of Viral Capsids

2007 ◽  
Vol 81 (20) ◽  
pp. 11363-11371 ◽  
Author(s):  
M. G. Lyman ◽  
B. Feierbach ◽  
D. Curanovic ◽  
M. Bisher ◽  
L. W. Enquist
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Sympathetic Neurons ◽  
Anterograde Transport ◽  
Viral Capsids ◽  
Green Fluorescent ◽  
Cultured Sympathetic Neurons ◽  
Retrograde Spread ◽  
Axonal Sorting

ABSTRACT Pseudorabies virus (PRV) mutants lacking the Us9 gene cannot spread from presynaptic to postsynaptic neurons in the rat visual system, although retrograde spread remains unaffected. We sought to recapitulate these findings in vitro using the isolator chamber system developed in our lab for analysis of the transneuronal spread of infection. The wild-type PRV Becker strain spreads efficiently to postsynaptic neurons in vitro, whereas the Us9-null strain does not. As determined by indirect immunofluorescence, the axons of Us9-null infected neurons do not contain the glycoproteins gB and gE, suggesting that their axonal sorting is dependent on Us9. Importantly, we failed to detect viral capsids in the axons of Us9-null infected neurons. We confirmed this observation by using three different techniques: by direct fluorescence of green fluorescent protein-tagged capsids; by transmission electron microscopy; and by live-cell imaging in cultured, sympathetic neurons. This finding has broad impact on two competing models for how virus particles are trafficked inside axons during anterograde transport and redefines a role for Us9 in viral sorting and transport.

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 Heterotrimeric Kinesin II Is the Microtubule Motor Protein Responsible for Pigment Dispersion in Xenopus Melanophores

1998 ◽  
Vol 143 (6) ◽  
pp. 1547-1558 ◽  
Author(s):  
M. Carolina Tuma ◽  
Andrew Zill ◽  
Nathalie Le Bot ◽  
Isabelle Vernos ◽  
Vladimir Gelfand
Keyword(s):  
Fluorescent Protein ◽  
Dominant Negative ◽  
Pigment Dispersion ◽  
Tail Region ◽  
Microtubule Motors ◽  
Microtubule Motor ◽  
Alkaline Conditions ◽  
Membrane Bound ◽  
Conventional Kinesin ◽  
Green Fluorescent

Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720–3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles.

Cytoplasmic dynein-associated structures move bidirectionallyin vivo

2002 ◽  
Vol 115 (7) ◽  
pp. 1453-1460 ◽  
Author(s):  
Shuo Ma ◽  
Rex L. Chisholm
Keyword(s):  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Anterograde Transport ◽  
Directional Movement ◽  
Motor Activities ◽  
Direction Of Movement ◽  
Linear Movement ◽  
Bidirectional Movement ◽  
Green Fluorescent

Intracellular organelle transport is driven by motors that act upon microtubules or microfilaments. The microtubulebased motors, cytoplasmic dynein and kinesin, are believed to be responsible for retrograde and anterograde transport of intracellular cargo along microtubules. Many vesicles display bidirectional movement; however, the mechanism regulating directionality is unresolved. Directional movement might be accomplished by alternative binding of different motility factors to the cargo. Alternatively,different motors could associate with the same cargo and have their motor activity regulated. Although several studies have focused on the behavior of specific types of cargoes, little is known about the traffic of the motors themselves and how it correlates with cargo movement. To address this question, we studied cytoplasmic dynein dynamics in living Dictyostelium cells expressing dynein intermediate chain-green fluorescent protein (IC-GFP) fusion in an IC-null background. Dynein-associated structures display fast linear movement along microtubules in both minus-end and plus-end directions, with velocities similar to that of dynein and kinesin-like motors. In addition, dynein puncta often rapidly reverse their direction. Dynein stably associates with cargo moving in both directions as well as with those that rapidly reverse their direction of movement, suggesting that directional movement is not regulated by altering motor-cargo association but rather by switching activity of motors associated with the cargo. These observations suggest that both plus- and minus-end-directed motors associate with a given cargo and that coordinated regulation of motor activities controls vesicle directionality.

scholarly journals An Ordered Inheritance Strategy for the Golgi Apparatus: Visualization of Mitotic Disassembly Reveals a Role for the Mitotic Spindle

1998 ◽  
Vol 141 (4) ◽  
pp. 955-966 ◽  
Author(s):  
David T. Shima ◽  
Noemí Cabrera-Poch ◽  
Rainer Pepperkok ◽  
Graham Warren
Keyword(s):  
Golgi Apparatus ◽  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Retrograde Transport ◽  
Early Prophase ◽  
Nuclear Envelope Breakdown ◽  
Microtubule Disruption ◽  
Low Copy Number ◽  
Er Exit Sites ◽  
Green Fluorescent

During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1–3-μm fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.

Melanopsin and non-melanopsin expressing retinal ganglion cells innervate the hypothalamic suprachiasmatic nucleus

2003 ◽  
Vol 20 (6) ◽  
pp. 601-610 ◽  
Author(s):  
PATRICIA J. SOLLARS ◽  
CYNTHIA A. SMERASKI ◽  
JESSICA D. KAUFMAN ◽  
MALCOLM D. OGILVIE ◽  
IGNACIO PROVENCIO ◽  
...  
Keyword(s):  
Retinal Ganglion Cells ◽  
Suprachiasmatic Nucleus ◽  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Ganglion Cells ◽  
Optic Tract ◽  
Retrograde Transport ◽  
Lateral Geniculate Body ◽  
Retinal Ganglion ◽  
Green Fluorescent

Retinal input to the hypothalamic suprachiasmatic nucleus (SCN) synchronizes the SCN circadian oscillator to the external day/night cycle. Retinal ganglion cells that innervate the SCN via the retinohypothalamic tract are intrinsically light sensitive and express melanopsin. In this study, we provide data indicating that not all SCN-projecting retinal ganglion cells express melanopsin. To determine the proportion of ganglion cells afferent to the SCN that express melanopsin, ganglion cells were labeled following transsynaptic retrograde transport of a recombinant of the Bartha strain of pseudorabies virus (PRV152) constructed to express the enhanced green fluorescent protein (EGFP). PRV152 injected into the anterior chamber of the eye retrogradely infects four retinorecipient nuclei in the brain via autonomic circuits to the eye, resulting in transneuronally labeled ganglion cells in the contralateral retina 96 h after intraocular infection. In animals with large bilateral lesions of the lateral geniculate body/optic tract, ganglion cells labeled with PRV152 are retrogradely infected from only the SCN. In these animals, most PRV152-infected ganglion cells were immunoreactive for melanopsin. However, a significant percentage (10–20%) of EGFP-labeled ganglion cells did not express melanopsin. These data suggest that in addition to the intrinsically light-sensitive melanopsin-expressing ganglion cells, conventional ganglion cells also innervate the SCN. Thus, it appears that the rod/cone system of photoreceptors may provide signals to the SCN circadian system independent of intrinsically light-sensitive melanopsin ganglion cells.

scholarly journals Spindle Dynamics during Meiosis in Drosophila Oocytes

1997 ◽  
Vol 137 (6) ◽  
pp. 1321-1336 ◽  
Author(s):  
Sharyn A. Endow ◽  
Donald J. Komma
Keyword(s):  
Fluorescent Protein ◽  
Meiotic Spindle ◽  
Oocyte Activation ◽  
New Information ◽  
Microtubule Motor ◽  
Meiotic Spindles ◽  
Green Fluorescent ◽  
Microtubule Motor Protein ◽  
Meiosis I

Mature oocytes of Drosophila are arrested in metaphase of meiosis I. Upon activation by ovulation or fertilization, oocytes undergo a series of rapid changes that have not been directly visualized previously. We report here the use of the Nonclaret disjunctional (Ncd) microtubule motor protein fused to the green fluorescent protein (GFP) to monitor changes in the meiotic spindle of live oocytes after activation in vitro. Meiotic spindles of metaphase-arrested oocytes are relatively stable, however, meiotic spindles of in vitro–activated oocytes are highly dynamic: the spindles elongate, rotate around their long axis, and undergo an acute pivoting movement to reorient perpendicular to the oocyte surface. Many oocytes spontaneously complete the meiotic divisions, permitting visualization of progression from meiosis I to II. The movements of the spindle after oocyte activation provide new information about the dynamic changes in the spindle that occur upon re-entry into meiosis and completion of the meiotic divisions. Spindles in live oocytes mutant for a lossof-function ncd allele fused to gfp were also imaged. The genesis of spindle defects in the live mutant oocytes provides new insights into the mechanism of Ncd function in the spindle during the meiotic divisions.

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