Aiming for invadopodia: organizing polarized delivery at sites of invasion

MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast. Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain. Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting. We also show that the actin cable–dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo. Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles. Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein. The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.

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Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of Madin-Darby canine kidney (MDCK) cells

Journal of Cell Science ◽

10.1242/jcs.103.3.677 ◽

1992 ◽

Vol 103 (3) ◽

pp. 677-687 ◽

Cited By ~ 4

Author(s):

G.K. Ojakian ◽

R. Schwimmer

Keyword(s):

Apical Membrane ◽

Mdck Cells ◽

Basolateral Membrane ◽

Immunogold Electron Microscopy ◽

Apical Plasma Membrane ◽

Apical Surface ◽

Transport Pathways ◽

Intracellular Targeting ◽

Polarized Delivery ◽

Membrane Recognition

Previous experiments on MDCK cells have demonstrated that the polarized appearance of a 135 kDa glycoprotein (gp135) on the apical plasma membrane can occur through the insertion of both newly synthesized gp135 as well as a pre-existing gp135 intracellular pool. In this study, anticytoskeletal drugs were utilized to determine the role of the cytoskeleton in the polarized delivery of gp135. Colchicine and nocodazole produced a 15–20% inhibition in the apical surface accumulation of newly synthesized gp135 and inhibited the appearance of the gp135 pool by approximately 33%, while cytochalasin D had no affect on the apical accumulation of either newly synthesized gp135 or the gp135 pool. These results indicate that microtubules, but not microfilaments, are involved in the intracellular targeting of gp135. Quantitative immunogold electron microscopy of nocodazole-treated cells demonstrated that gp135 was not mistargeted to the basolateral membrane, suggesting the possibility that some vesicles containing gp135 did not fuse with the apical membrane and remained in the cells. These experiments demonstrate that microtubules are an important component of gp135 insertion into the apical membrane. They also suggest that gp135 resides within vesicles which have an apical membrane recognition signal and cannot fuse with the basolateral membrane. The possibility that these data, and those of others, could support a hypothesis for the presence of two constitutive apical transport pathways is discussed.

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Reversed polarized delivery of an aquaporin-2 mutant causes dominant nephrogenic diabetes insipidus

The Journal of Cell Biology ◽

10.1083/jcb.200309017 ◽

2003 ◽

Vol 163 (5) ◽

pp. 1099-1109 ◽

Cited By ~ 92

Author(s):

Erik-Jan Kamsteeg ◽

Daniel G. Bichet ◽

Irene B.M. Konings ◽

Hubert Nivet ◽

Michelle Lonergan ◽

...

Keyword(s):

Diabetes Insipidus ◽

Water Conservation ◽

Nephrogenic Diabetes Insipidus ◽

Water Channel ◽

Apical Plasma Membrane ◽

Apical Surface ◽

Aquaporin 2 ◽

Frame Shift ◽

Polarized Delivery ◽

Mutant Forms

Vasopressin regulates body water conservation by redistributing aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical surface of renal collecting ducts, resulting in water reabsorption from urine. Mutations in AQP2 cause autosomal nephrogenic diabetes insipidus (NDI), a disease characterized by the inability to concentrate urine. Here, we report a frame-shift mutation in AQP2 causing dominant NDI. This AQP2 mutant is a functional water channel when expressed in Xenopus oocytes. However, expressed in polarized renal cells, it is misrouted to the basolateral instead of apical plasma membrane. Additionally, this mutant forms heterotetramers with wild-type AQP2 and redirects this complex to the basolateral surface. The frame shift induces a change in the COOH terminus of AQP2, creating both a leucine- and a tyrosine-based motif, which cause the reversed sorting of AQP2. Our data reveal a novel cellular phenotype in dominant NDI and show that dominance of basolateral sorting motifs in a mutant subunit can be the molecular basis for disease.

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Sec2p Mediates Nucleotide Exchange on Sec4p and Is Involved in Polarized Delivery of Post-Golgi Vesicles

The Journal of Cell Biology ◽

10.1083/jcb.137.7.1495 ◽

1997 ◽

Vol 137 (7) ◽

pp. 1495-1509 ◽

Cited By ~ 234

Author(s):

Christiane Walch-Solimena ◽

Ruth N. Collins ◽

Peter J. Novick

Keyword(s):

Secretory Pathway ◽

Essential Gene ◽

Vesicular Transport ◽

Small Gtpase ◽

Nucleotide Exchange ◽

Present Evidence ◽

Golgi Vesicles ◽

Polarized Delivery ◽

Exchange Protein

The small GTPase Sec4p is required for vesicular transport at the post-Golgi stage of yeast secretion. Here we present evidence that mutations in SEC2, itself an essential gene that acts at the same stage of the secretory pathway, cause Sec4p to mislocalize as a result of a random rather than a polarized accumulation of vesicles. Sec2p and Sec4p interact directly, with the nucleotide-free conformation of Sec4p being the preferred state for interaction with Sec2p. Sec2p functions as an exchange protein, catalyzing the dissociation of GDP from Sec4 and promoting the binding of GTP. We propose that Sec2p functions to couple the activation of Sec4p to the polarized delivery of vesicles to the site of exocytosis.

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A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin

Trends in Cell Biology ◽

10.1016/0962-8924(91)90103-g ◽

1991 ◽

Vol 1 (5) ◽

pp. 113

Author(s):

C.B. Brewer ◽

M.G. Roth

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Amino Acid Change ◽

Cytoplasmic Domain ◽

Single Amino Acid ◽

Influenza Virus Hemagglutinin ◽

Single Amino Acid Change ◽

Polarized Delivery

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Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization

Molecular Biology of the Cell ◽

10.1091/mbc.e13-02-0078 ◽

2013 ◽

Vol 24 (12) ◽

pp. 1996-2007 ◽

Cited By ~ 16

Author(s):

Robert T. Youker ◽

Jennifer R. Bruns ◽

Simone A. Costa ◽

Youssef Rbaibi ◽

Frederick Lanni ◽

...

Keyword(s):

Fluorescent Protein ◽

Higher Order ◽

Neurotrophin Receptor ◽

Protein Oligomerization ◽

Apical Surface ◽

Sorting Signals ◽

Stalk Domain ◽

Polarized Delivery ◽

Apical Sorting

The sorting signals that direct proteins to the apical surface of polarized epithelial cells are complex and can include posttranslational modifications, such as N- and O-linked glycosylation. Efficient apical sorting of the neurotrophin receptor p75 is dependent on its O-glycosylated membrane proximal stalk, but how this domain mediates targeting is unknown. Protein oligomerization or clustering has been suggested as a common step in the segregation of all apical proteins. Like many apical proteins, p75 forms dimers, and we hypothesized that formation of higher-order clusters mediated by p75 dimerization and interactions of the stalk facilitate its apical sorting. Using fluorescence fluctuation techniques (photon-counting histogram and number and brightness analyses) to study p75 oligomerization status in vivo, we found that wild-type p75–green fluorescent protein forms clusters in the trans-Golgi network (TGN) but not at the plasma membrane. Disruption of either the dimerization motif or the stalk domain impaired both clustering and polarized delivery. Manipulation of O-glycan processing or depletion of multiple galectins expressed in Madin-Darby canine kidney cells had no effect on p75 sorting, suggesting that the stalk domain functions as a structural prop to position other determinants in the lumenal domain of p75 for oligomerization. Additionally, a p75 mutant with intact dimerization and stalk motifs but with a dominant basolateral sorting determinant (Δ250 mutant) did not form oligomers, consistent with a requirement for clustering in apical sorting. Artificially enhancing dimerization restored clustering to the Δ250 mutant but was insufficient to reroute this mutant to the apical surface. Together these studies demonstrate that clustering in the TGN is required for normal biosynthetic apical sorting of p75 but is not by itself sufficient to reroute a protein to the apical surface in the presence of a strong basolateral sorting determinant. Our studies shed new light on the hierarchy of polarized sorting signals and on the mechanisms by which newly synthesized proteins are segregated in the TGN for eventual apical delivery.

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A Rab8-specific GDP/GTP Exchange Factor Is Involved in Actin Remodeling and Polarized Membrane Transport

Molecular Biology of the Cell ◽

10.1091/mbc.e02-03-0143 ◽

2002 ◽

Vol 13 (9) ◽

pp. 3268-3280 ◽

Cited By ~ 143

Author(s):

Katarina Hattula ◽

Johanna Furuhjelm ◽

Airi Arffman ◽

Johan Peränen

Keyword(s):

Cell Surface ◽

Phorbol Esters ◽

Outer Edge ◽

Dominant Negative ◽

Nucleotide Exchange ◽

Cortical Actin ◽

Animal Cells ◽

Dynamic Cell ◽

Polarized Delivery ◽

Surface Domains

The mechanisms mediating polarized delivery of vesicles to cell surface domains are poorly understood in animal cells. We have previously shown that expression of Rab8 promotes the formation of new cell surface domains through reorganization of actin and microtubules. To unravel the function of Rab8, we used the yeast two-hybrid system to search for potential Rab8-specific activators. We identified a coil-coiled protein (Rabin8), homologous to the rat Rabin3 that stimulated nucleotide exchange on Rab8 but not on Rab3A and Rab5. Furthermore, we show that rat Rabin3 has exchange activity on Rab8 but not on Rab3A, supporting the view that rat Rabin3 is the rat equivalent of human Rabin8. Rabin8 localized to the cortical actin and expression of Rabin8 resulted in remodeling of actin and the formation of polarized cell surface domains. Activation of PKC by phorbol esters enhanced translocation of both Rabin8 and Rab8-specific vesicles to the outer edge of lamellipodial structures. Moreover, coexpression of Rabin8 with dominant negative Rab8 (T22N) redistributes Rabin8 from cortical actin to Rab8-specific vesicles and promotes their polarized transport to cell protrusions. The C-terminal region of Rabin8 plays an essential role in this transport. We propose that Rabin8 is a Rab8-specific activator that is connected to processes that mediate polarized membrane traffic to dynamic cell surface structures.

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