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

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.

Download Full-text

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

Molecular Biology of the Cell ◽

10.1091/mbc.e07-03-0261 ◽

2008 ◽

Vol 19 (1) ◽

pp. 274-283 ◽

Cited By ~ 117

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.

Download Full-text

Two Heteromeric Kinesin Complexes in Chemosensory Neurons and Sensory Cilia of Caenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.10.2.345 ◽

1999 ◽

Vol 10 (2) ◽

pp. 345-360 ◽

Cited By ~ 105

Author(s):

Dawn Signor ◽

Karen P. Wedaman ◽

Lesilee S. Rose ◽

Jonathan M. Scholey

Keyword(s):

Signal Transduction ◽

Fluorescent Protein ◽

Partial Purification ◽

Localization Pattern ◽

Chemosensory Neurons ◽

C Elegans ◽

Microtubule Motors ◽

Sensory Cilia ◽

Green Fluorescent ◽

Kinesin Family

Chemosensation in the nervous system of the nematodeCaenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia.

Download Full-text

Differential Localization of Rho Gtpases in Live Cells

The Journal of Cell Biology ◽

10.1083/jcb.152.1.111 ◽

2001 ◽

Vol 152 (1) ◽

pp. 111-126 ◽

Cited By ~ 472

Author(s):

David Michaelson ◽

Joseph Silletti ◽

Gretchen Murphy ◽

Peter D'Eustachio ◽

Mark Rush ◽

...

Keyword(s):

Rho Gtpases ◽

Fluorescent Protein ◽

Essential Feature ◽

Hypervariable Region ◽

Dominant Negative ◽

Live Cells ◽

Rho Proteins ◽

Guanine Nucleotide Dissociation Inhibitor ◽

Membrane Compartment ◽

Green Fluorescent

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

Download Full-text

Functional Analyses of a Putative, Membrane-Bound, Peroxisomal Protein Import Mechanism from the Apicomplexan Protozoan Toxoplasma gondii

Genes ◽

10.3390/genes9090434 ◽

2018 ◽

Vol 9 (9) ◽

pp. 434

Author(s):

Alison Mbekeani ◽

Will Stanley ◽

Vishal Kalel ◽

Noa Dahan ◽

Einat Zalckvar ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Protein Import ◽

Fluorescent Protein ◽

Metabolic Energy ◽

Peroxisomal Protein ◽

Genes Encoding ◽

Peroxisomal Targeting ◽

Membrane Bound ◽

Green Fluorescent

Peroxisomes are central to eukaryotic metabolism, including the oxidation of fatty acids—which subsequently provide an important source of metabolic energy—and in the biosynthesis of cholesterol and plasmalogens. However, the presence and nature of peroxisomes in the parasitic apicomplexan protozoa remains controversial. A survey of the available genomes revealed that genes encoding peroxisome biogenesis factors, so-called peroxins (Pex), are only present in a subset of these parasites, the coccidia. The basic principle of peroxisomal protein import is evolutionarily conserved, proteins harbouring a peroxisomal-targeting signal 1 (PTS1) interact in the cytosol with the shuttling receptor Pex5 and are then imported into the peroxisome via the membrane-bound protein complex formed by Pex13 and Pex14. Surprisingly, whilst Pex5 is clearly identifiable, Pex13 and, perhaps, Pex14 are apparently absent from the coccidian genomes. To investigate the functionality of the PTS1 import mechanism in these parasites, expression of Pex5 from the model coccidian Toxoplasma gondii was shown to rescue the import defect of Pex5-deleted Saccharomyces cerevisiae. In support of these data, green fluorescent protein (GFP) bearing the enhanced (e)PTS1 known to efficiently localise to peroxisomes in yeast, localised to peroxisome-like bodies when expressed in Toxoplasma. Furthermore, the PTS1-binding domain of Pex5 and a PTS1 ligand from the putatively peroxisome-localised Toxoplasma sterol carrier protein (SCP2) were shown to interact in vitro. Taken together, these data demonstrate that the Pex5–PTS1 interaction is functional in the coccidia and indicate that a nonconventional peroxisomal import mechanism may operate in the absence of Pex13 and Pex14.

Download Full-text

Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

The Journal of Cell Biology ◽

10.1083/jcb.143.6.1505 ◽

1998 ◽

Vol 143 (6) ◽

pp. 1505-1521 ◽

Cited By ~ 245

Author(s):

Brian Storrie ◽

Jamie White ◽

Sabine Röttger ◽

Ernst H.K. Stelzer ◽

Tatsuo Suganuma ◽

...

Keyword(s):

Fluorescent Protein ◽

Time Lapse ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Protein Synthesis Inhibitors ◽

Microtubule Depolymerization ◽

Golgi Stack ◽

Gradual Accumulation ◽

Green Fluorescent ◽

Er Export

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export. Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.

Download Full-text

Heterologous downregulation of vasopressin type 2 receptor is induced by transferrin

AJP Renal Physiology ◽

10.1152/ajprenal.00438.2011 ◽

2013 ◽

Vol 304 (5) ◽

pp. F553-F564 ◽

Cited By ~ 3

Author(s):

Richard Bouley ◽

Paula Nunes ◽

Billy Andriopoulos ◽

Margaret McLaughlin ◽

Matthew J. Webber ◽

...

Keyword(s):

Fluorescent Protein ◽

Dominant Negative ◽

Target Cells ◽

Intracellular Camp ◽

Coated Pits ◽

Parathyroid Hormone Receptor ◽

Body Fluid Homeostasis ◽

Green Fluorescent ◽

G Protein Coupled

Vasopressin (VP) binds to the vasopressin type 2 receptor (V2R) to trigger physiological effects including body fluid homeostasis and blood pressure regulation. Signaling is terminated by receptor downregulation involving clathrin-mediated endocytosis and V2R degradation. We report here that both native and epitope-tagged V2R are internalized from the plasma membrane of LLC-PK1 kidney epithelial cells in the presence of another ligand, transferrin (Tf). The presence of iron-saturated Tf (holo-Tf; 4 h) reduced V2R binding sites at the cell surface by up to 33% while iron-free (apo-Tf) had no effect. However, no change in green fluorescent protein-tagged V2R distribution was observed in the presence of bovine serum albumin, atrial natriuretic peptide, or ANG II. Conversely, holo-Tf did not induce the internalization of another G protein-coupled receptor, the parathyroid hormone receptor. In contrast to the effect of VP, Tf did not increase intracellular cAMP or modify aquaporin-2 distribution in these cells, although addition of VP and Tf together augmented VP-induced V2R internalization. Tf receptor coimmunoprecipitated with V2R, suggesting that they interact closely, which may explain the additive effect of VP and Tf on V2R endocytosis. Furthermore, Tf-induced V2R internalization was abolished in cells expressing a dominant negative dynamin (K44A) mutant, indicating the involvement of clathrin-coated pits. We conclude that Tf can induce heterologous downregulation of the V2R and this might desensitize VP target cells without activating downstream V2R signaling events. It also provides new insights into urine-concentrating defects observed in rat models of hemochromatosis.

Download Full-text

Intervention of Bro1 in pH-Responsive Rim20 Localization in Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00027-10 ◽

2010 ◽

Vol 9 (4) ◽

pp. 532-538 ◽

Cited By ~ 5

Author(s):

Jacob H. Boysen ◽

Shoba Subramanian ◽

Aaron P. Mitchell

Keyword(s):

Target Genes ◽

Fluorescent Protein ◽

Growth Conditions ◽

Yeast Cells ◽

Endosomal Trafficking ◽

The Novel ◽

Pathway Activation ◽

Alkaline Conditions ◽

Acidic Conditions ◽

Green Fluorescent

ABSTRACT Yeast cells contain two Bro1 domain proteins: Bro1, which is required for endosomal trafficking, and Rim20, which is required for the response to the external pH via the Rim101 pathway. Rim20 associates with endosomal structures under alkaline growth conditions, when it promotes activation of Rim101 through proteolytic cleavage. We report here that the pH-dependent localization of Rim20 is contingent on the amount of Bro1 in the cell. Cells that lack Bro1 have increased endosomal Rim20-green fluorescent protein (GFP) under acidic conditions; cells that overexpress Bro1 have reduced endosomal Rim20-GFP under acidic or alkaline conditions. The novel endosomal association of Rim20-GFP in the absence of Bro1 requires ESCRT components including Vps27 but not specific Rim101 pathway components such as Dfg16. Vps27 influences the localization of Bro1 but is not required for RIM101 pathway activation in wild-type cells, thus suggesting that Rim20 enters the Bro1 localization pathway when a vacancy exists. Despite altered localization of Rim20, the lack of Bro1 does not bypass the need for signaling protein Dfg16 to activate Rim101, as evidenced by the expression levels of the Rim101 target genes RIM8 and SMP1. Therefore, endosomal association of Rim20 is not sufficient to promote Rim101 activation.

Download Full-text

Smooth muscle cell-specific transcription is regulated by nuclear localization of the myocardin-related transcription factors

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00864.2006 ◽

2007 ◽

Vol 292 (2) ◽

pp. H1170-H1180 ◽

Cited By ~ 69

Author(s):

Jeremiah S. Hinson ◽

Matthew D. Medlin ◽

Kashelle Lockman ◽

Joan M. Taylor ◽

Christopher P. Mack

Keyword(s):

Smooth Muscle ◽

Transcription Factors ◽

Nuclear Localization ◽

Transforming Growth Factor ◽

Serum Response Factor ◽

Fluorescent Protein ◽

Nuclear Translocation ◽

Dominant Negative ◽

Specific Gene ◽

Green Fluorescent

On the basis of our previous studies on RhoA signaling in smooth muscle cells (SMC), we hypothesized that RhoA-mediated nuclear translocalization of the myocardin-related transcription factors (MRTFs) was important for regulating SMC phenotype. MRTF-A protein and MRTF-B message were detected in aortic SMC and in many adult mouse organs that contain a large SMC component. Both MRTFs upregulated SMC-specific promoter activity as well as endogenous SM22α expression in multipotential 10T1/2 cells, although to a lesser extent than myocardin. We used enhanced green fluorescent protein (EGFP) fusion proteins to demonstrate that the myocardin factors have dramatically different localization patterns and that the stimulation of SMC-specific transcription by certain RhoA-dependent agonists was likely mediated by increased nuclear translocation of the MRTFs. Importantly, a dominant-negative form of MRTF-A (ΔB1/B2) that traps endogenous MRTFs in the cytoplasm inhibited the SM α-actin, SM22α, and SM myosin heavy chain promoters in SMC and attenuated the effects of sphingosine 1-phosphate and transforming growth factor (TGF)-β on SMC-specific transcription. Our data confirmed the importance of the NH2-terminal RPEL domains for regulating MRTF localization, but our analysis of MRTF-A/myocardin chimeras and myocardin RPEL2 mutations indicated that the myocardin B1/B2 region can override this signal. Gel shift assays demonstrated that myocardin factor activity correlated well with ternary complex formation at the SM α-actin CArGs and that MRTF-serum response factor interactions were partially dependent on CArG sequence. Taken together, our results indicate that the MRTFs regulate SMC-specific gene expression in at least some SMC subtypes and that regulation of MRTF nuclear localization may be important for the effects of selected agonists on SMC phenotype.

Download Full-text

Myosin-V, Kinesin-1, and Kinesin-3 Cooperate in Hyphal Growth of the FungusUstilago maydis

Molecular Biology of the Cell ◽

10.1091/mbc.e05-04-0272 ◽

2005 ◽

Vol 16 (11) ◽

pp. 5191-5201 ◽

Cited By ~ 83

Author(s):

Isabel Schuchardt ◽

Daniela Aßmann ◽

Eckhard Thines ◽

Christian Schuberth ◽

Gero Steinberg

Keyword(s):

Fluorescent Protein ◽

Hyphal Growth ◽

Myosin V ◽

Long Distance ◽

Long Distance Transport ◽

Conventional Kinesin ◽

Green Fluorescent ◽

Mutant Cells ◽

Distance Transport

Long-distance transport is crucial for polar-growing cells, such as neurons and fungal hyphae. Kinesins and myosins participate in this process, but their functional interplay is poorly understood. Here, we investigate the role of kinesin motors in hyphal growth of the plant pathogen Ustilago maydis. Although the microtubule plus-ends are directed to the hyphal tip, of all 10 kinesins analyzed, only conventional kinesin (Kinesin-1) and Unc104/Kif1A-like kinesin (Kinesin-3) were up-regulated in hyphae and they are essential for extended hyphal growth. Δkin1 and Δkin3 mutant hyphae grew irregular and remained short, but they were still able to grow polarized. No additional phenotype was detected in Δkin1rkin3 double mutants, but polarity was lost in Δmyo5rkin1 and Δmyo5rkin3 mutant cells, suggesting that kinesins and class V myosin cooperate in hyphal growth. Consistent with such a role in secretion, fusion proteins of green fluorescent protein and Kinesin-1, Myosin-V, and Kinesin-3 accumulate in the apex of hyphae, a region where secretory vesicles cluster to form the fungal Spitzenkörper. Quantitative assays revealed a role of Kin3 in secretion of acid phosphatase, whereas Kin1 was not involved. Our data demonstrate that just two kinesins and at least one myosin support hyphal growth.

Download Full-text

Lamin A/C Binding Protein LAP2α Is Required for Nuclear Anchorage of Retinoblastoma Protein

Molecular Biology of the Cell ◽

10.1091/mbc.e02-07-0450 ◽

2002 ◽

Vol 13 (12) ◽

pp. 4401-4413 ◽

Cited By ~ 169

Author(s):

Ewa Markiewicz ◽

Thomas Dechat ◽

Roland Foisner ◽

Roy. A Quinlan ◽

Christopher J. Hutchison

Keyword(s):

Tissue Culture ◽

Fluorescent Protein ◽

Solid Phase ◽

Retinoblastoma Protein ◽

Dominant Negative ◽

Lamin A ◽

Dependent Manner ◽

Culture Cells ◽

Tissue Culture Cells ◽

Green Fluorescent

The phosphorylation-dependent anchorage of retinoblastoma protein Rb in the nucleus is essential for its function. We show that its pocket C domain is both necessary and sufficient for nuclear anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays using glutathioneS-transferase-fusion of Rb pockets A, B, and C revealed a direct association of lamin C exclusively to pocket C. Lamina-associated polypeptide (LAP) 2α, a binding partner of lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2α was immunoprecipitated from soluble nuclear fractions, lamins A/C and hypophosphorylated Rb were coprecipitated efficiently. Similarly, immunoprecipitation of expressed GFP-Rb fragments by using anti-GFP antibodies coprecipitated LAP2α, provided that pocket C was present in the GFP chimeras. On redistribution of endogenous lamin A/C and LAP2α into nuclear aggregates by overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered into these aggregates. In primary skin fibroblasts, LAP2α is expressed in a growth-dependent manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the absence of LAP2α. Together, these data suggest that hypophosphorylated Rb is anchored in the nucleus by the interaction of pocket C with LAP2α–lamin A/C complexes.

Download Full-text