Journal of Cell Biology

The enterocyte microvillus is a vesicle-generating organelle.

Publication Date: 2009 Jun 29 PMID: 19564407
Authors: McConnell, R. E. - Higginbotham, J. N. - Shifrin, D. A. Jr - Tabb, D. L. - Coffey, R. J. - Tyska, M. J.
Journal: J Cell Biol

For decades, enterocyte brush border microvilli have been viewed as passive cytoskeletal scaffolds that serve to increase apical membrane surface area. However, recent studies revealed that in the in vitro context of isolated brush borders, myosin-1a (myo1a) powers the sliding of microvillar membrane along core actin bundles. This activity also leads to the shedding of small vesicles from microvillar tips, suggesting that microvilli may function as vesicle-generating organelles in vivo. In this study, we present data in support of this hypothesis, showing that enterocyte microvilli release unilamellar vesicles into the intestinal lumen; these vesicles retain the right side out orientation of microvillar membrane, contain catalytically active brush border enzymes, and are specifically enriched in intestinal alkaline phosphatase. Moreover, myo1a knockout mice demonstrate striking perturbations in vesicle production, clearly implicating this motor in the in vivo regulation of this novel activity. In combination, these data show that microvilli function as vesicle-generating organelles, which enable enterocytes to deploy catalytic activities into the intestinal lumen.

post to: CiteULike

Demonstration of catch bonds between an integrin and its ligand.

Publication Date: 2009 Jun 29 PMID: 19564406
Authors: Kong, F. - Garcia, A. J. - Mould, A. P. - Humphries, M. J. - Zhu, C.
Journal: J Cell Biol

Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. How force regulates integrin-ligand dissociation is unclear. We used atomic force microscopy to measure the force-dependent lifetimes of single bonds between a fibronectin fragment and an integrin alpha(5)beta(1)-Fc fusion protein or membrane alpha(5)beta(1). Force prolonged bond lifetimes in the 10-30-pN range, a counterintuitive behavior called catch bonds. Changing cations from Ca(2+)/Mg(2+) to Mg(2+)/EGTA and to Mn(2+) caused longer lifetime in the same 10-30-pN catch bond region. A truncated alpha(5)beta(1) construct containing the headpiece but not the legs formed longer-lived catch bonds that were not affected by cation changes at forces <30 pN. Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range. Thus, catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension.

post to: CiteULike

A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling.

Publication Date: 2009 Jun 29 PMID: 19564405
Authors: Frohlich, F. - Moreira, K. - Aguilar, P. S. - Hubner, N. C. - Mann, M. - Walter, P. - Walther, T. C.
Journal: J Cell Biol

The protein and lipid composition of eukaryotic plasma membranes is highly dynamic and regulated according to need. The sphingolipid-responsive Pkh kinases are candidates for mediating parts of this regulation, as they affect a diverse set of plasma membrane functions, such as cortical actin patch organization, efficient endocytosis, and eisosome assembly. Eisosomes are large protein complexes underlying the plasma membrane and help to sort a group of membrane proteins into distinct domains. In this study, we identify Nce102 in a genome-wide screen for genes involved in eisosome organization and Pkh kinase signaling. Nce102 accumulates in membrane domains at eisosomes where Pkh kinases also localize. The relative abundance of Nce102 in these domains compared with the rest of the plasma membrane is dynamically regulated by sphingolipids. Furthermore, Nce102 inhibits Pkh kinase signaling and is required for plasma membrane organization. Therefore, Nce102 might act as a sensor of sphingolipids that regulates plasma membrane function.

post to: CiteULike

Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning.

Publication Date: 2009 Jun 29 PMID: 19564404
Authors: Rocha, N. - Kuijl, C. - van der Kant, R. - Janssen, L. - Houben, D. - Janssen, H. - Zwart, W. - Neefjes, J.
Journal: J Cell Biol

Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150(Glued) bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)-LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7-RILP complex to remove p150(Glued) and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.

post to: CiteULike

Biogenesis of cytochrome b6 in photosynthetic membranes.

Publication Date: 2009 Jun 29 PMID: 19564403
Authors: Saint-Marcoux, D. - Wollman, F. A. - de Vitry, C.
Journal: J Cell Biol

In chloroplasts, binding of a c'-heme to cytochrome b(6) on the stromal side of the thylakoid membranes requires a specific mechanism distinct from the one at work for c-heme binding to cytochromes f and c(6) on the lumenal side of membranes. Here, we show that the major protein components of this pathway, the CCBs, are bona fide transmembrane proteins. We demonstrate their association in a series of hetero-oligomeric complexes, some of which interact transiently with cytochrome b(6) in the process of heme delivery to the apoprotein. In addition, we provide preliminary evidence for functional assembly of cytochrome b(6)f complexes even in the absence of c'-heme binding to cytochrome b(6). Finally, we present a sequential model for apo- to holo-cytochrome b(6) maturation integrated within the assembly pathway of b(6)f complexes in the thylakoid membranes.

post to: CiteULike

Distinct cytoplasmic maturation steps of 40S ribosomal subunit precursors require hRio2.

Publication Date: 2009 Jun 29 PMID: 19564402
Authors: Zemp, I. - Wild, T. - O'Donohue, M. F. - Wandrey, F. - Widmann, B. - Gleizes, P. E. - Kutay, U.
Journal: J Cell Biol

During their biogenesis, 40S ribosomal subunit precursors are exported from the nucleus to the cytoplasm, where final maturation occurs. In this study, we show that the protein kinase human Rio2 (hRio2) is part of a late 40S preribosomal particle in human cells. Using a novel 40S biogenesis and export assay, we analyzed the contribution of hRio2 to late 40S maturation. Although hRio2 is not absolutely required for pre-40S export, deletion of its binding site for the export receptor CRM1 decelerated the kinetics of this process. Moreover, in the absence of hRio2, final cytoplasmic 40S maturation is blocked because the recycling of several trans-acting factors and cytoplasmic 18S-E precursor ribosomal RNA (rRNA [pre-rRNA]) processing are defective. Intriguingly, the physical presence of hRio2 but not its kinase activity is necessary for the release of hEnp1 from cytoplasmic 40S precursors. In contrast, hRio2 kinase activity is essential for the recycling of hDim2, hLtv1, and hNob1 as well as for 18S-E pre-rRNA processing. Thus, hRio2 is involved in late 40S maturation at several distinct steps.

post to: CiteULike

Motor-dependent microtubule disassembly driven by tubulin tyrosination.

Publication Date: 2009 Jun 29 PMID: 19564401
Authors: Peris, L. - Wagenbach, M. - Lafanechere, L. - Brocard, J. - Moore, A. T. - Kozielski, F. - Job, D. - Wordeman, L. - Andrieux, A.
Journal: J Cell Biol

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of alpha-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)-inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.

post to: CiteULike

Accuracy and precision in quantitative fluorescence microscopy.

Publication Date: 2009 Jun 29 PMID: 19564400
Authors: Waters, J. C.
Journal: J Cell Biol

The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Forster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.

post to: CiteULike

Antonio Giraldez: at the tip of the microRNA iceberg.

Publication Date: 2009 Jun 29 PMID: 19564399
Authors: Giraldez, A.
Journal: J Cell Biol

post to: CiteULike

The Drosophila deoxyhypusine hydroxylase homologue nero and its target eIF5A are required for cell growth and the regulation of autophagy.

Publication Date: 2009 Jun 29 PMID: 19546244
Authors: Patel, P. H. - Costa-Mattioli, M. - Schulze, K. L. - Bellen, H. J.
Journal: J Cell Biol

Hypusination is a unique posttranslational modification by which lysine is transformed into the atypical amino acid hypusine. eIF5A (eukaryotic initiation factor 5A) is the only known protein to contain hypusine. In this study, we describe the identification and characterization of nero, the Drosophila melanogaster deoxyhypusine hydroxylase (DOHH) homologue. nero mutations affect cell and organ size, bromodeoxyuridine incorporation, and autophagy. Knockdown of the hypusination target eIF5A via RNA interference causes phenotypes similar to nero mutations. However, loss of nero appears to cause milder phenotypes than loss of eIF5A. This is partially explained through a potential compensatory mechanism by which nero mutant cells up-regulate eIF5A levels. The failure of eIF5A up-regulation to rescue nero mutant phenotypes suggests that hypusination is required for eIF5A function. Furthermore, expression of enzymatically impaired forms of DOHH fails to rescue nero clones, indicating that hypusination activity is important for nero function. Our data also indicate that nero and eIF5A are required for cell growth and affect autophagy and protein synthesis.

post to: CiteULike

Desmoglein 1-dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis.

Publication Date: 2009 Jun 29 PMID: 19546243
Authors: Getsios, S. - Simpson, C. L. - Kojima, S. - Harmon, R. - Sheu, L. J. - Dusek, R. L. - Cornwell, M. - Green, K. J.
Journal: J Cell Biol

Dsg1 (desmoglein 1) is a member of the cadherin family of Ca(2+)-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal growth factor receptor-Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.

post to: CiteULike

MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes.

Publication Date: 2009 Jun 29 PMID: 19546242
Authors: Bastiani, M. - Liu, L. - Hill, M. M. - Jedrychowski, M. P. - Nixon, S. J. - Lo, H. P. - Abankwa, D. - Luetterforst, R. - Fernandez-Rojo, M. - Breen, M. R. - Gygi, S. P. - Vinten, J. - Walser, P. J. - North, K. N. - Hancock, J. F. - Pilch, P. F. - Parton, R. G.
Journal: J Cell Biol

Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer-based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein.

post to: CiteULike

Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1.

Publication Date: 2009 Jun 29 PMID: 19546241
Authors: Tibelius, A. - Marhold, J. - Zentgraf, H. - Heilig, C. E. - Neitzel, H. - Ducommun, B. - Rauch, A. - Ho, A. D. - Bartek, J. - Kramer, A.
Journal: J Cell Biol

Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life. Although primary microcephaly can be caused by mutations in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling. In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently deregulated activation of centrosomal cyclin B-Cdk1.

post to: CiteULike

HUMMR, a hypoxia- and HIF-1alpha-inducible protein, alters mitochondrial distribution and transport.

Publication Date: 2009 Jun 15 PMID: 19528298
Authors: Li, Y. - Lim, S. - Hoffman, D. - Aspenstrom, P. - Federoff, H. J. - Rempe, D. A.
Journal: J Cell Biol

Mitochondrial transport is critical for maintenance of normal neuronal function. Here, we identify a novel mitochondria protein, hypoxia up-regulated mitochondrial movement regulator (HUMMR), which is expressed in neurons and is markedly induced by hypoxia-inducible factor 1 alpha (HIF-1alpha). Interestingly, HUMMR interacts with Miro-1 and Miro-2, mitochondrial proteins that are critical for mediating mitochondrial transport. Interestingly, knockdown of HUMMR or HIF-1 function in neurons exposed to hypoxia markedly reduces mitochondrial content in axons. Because mitochondrial transport and distribution are inextricably linked, the impact of reduced HUMMR function on the direction of mitochondrial transport was also explored. Loss of HUMMR function in hypoxia diminished the percentage of motile mitochondria moving in the anterograde direction and enhanced the percentage moving in the retrograde direction. Thus, HUMMR, a novel mitochondrial protein induced by HIF-1 and hypoxia, biases mitochondria transport in the anterograde direction. These findings have broad implications for maintenance of neuronal viability and function during physiological and pathological states.

post to: CiteULike

Formation of cristae and crista junctions in mitochondria depends on antagonism between Fcj1 and Su e/g.

Publication Date: 2009 Jun 15 PMID: 19528297
Authors: Rabl, R. - Soubannier, V. - Scholz, R. - Vogel, F. - Mendl, N. - Vasiljev-Neumeyer, A. - Korner, C. - Jagasia, R. - Keil, T. - Baumeister, W. - Cyrklaff, M. - Neupert, W. - Reichert, A. S.
Journal: J Cell Biol

Crista junctions (CJs) are important for mitochondrial organization and function, but the molecular basis of their formation and architecture is obscure. We have identified and characterized a mitochondrial membrane protein in yeast, Fcj1 (formation of CJ protein 1), which is specifically enriched in CJs. Cells lacking Fcj1 lack CJs, exhibit concentric stacks of inner membrane in the mitochondrial matrix, and show increased levels of F(1)F(O)-ATP synthase (F(1)F(O)) supercomplexes. Overexpression of Fcj1 leads to increased CJ formation, branching of cristae, enlargement of CJ diameter, and reduced levels of F(1)F(O) supercomplexes. Impairment of F(1)F(O) oligomer formation by deletion of its subunits e/g (Su e/g) causes CJ diameter enlargement and reduction of cristae tip numbers and promotes cristae branching. Fcj1 and Su e/g genetically interact. We propose a model in which the antagonism between Fcj1 and Su e/g locally modulates the F(1)F(O) oligomeric state, thereby controlling membrane curvature of cristae to generate CJs and cristae tips.

post to: CiteULike

Role of Inn1 and its interactions with Hof1 and Cyk3 in promoting cleavage furrow and septum formation in S. cerevisiae.

Publication Date: 2009 Jun 15 PMID: 19528296
Authors: Nishihama, R. - Schreiter, J. H. - Onishi, M. - Vallen, E. A. - Hanna, J. - Moravcevic, K. - Lippincott, M. F. - Han, H. - Lemmon, M. A. - Pringle, J. R. - Bi, E.
Journal: J Cell Biol

Cytokinesis requires coordination of actomyosin ring (AMR) contraction with rearrangements of the plasma membrane and extracellular matrix. In Saccharomyces cerevisiae, new membrane, the chitin synthase Chs2 (which forms the primary septum [PS]), and the protein Inn1 are all delivered to the division site upon mitotic exit even when the AMR is absent. Inn1 is essential for PS formation but not for Chs2 localization. The Inn1 C-terminal region is necessary for localization, and distinct PXXP motifs in this region mediate functionally important interactions with SH3 domains in the cytokinesis proteins Hof1 (an F-BAR protein) and Cyk3 (whose overexpression can restore PS formation in inn1Delta cells). The Inn1 N terminus resembles C2 domains but does not appear to bind phospholipids; nonetheless, when overexpressed or fused to Hof1, it can provide Inn1 function even in the absence of the AMR. Thus, Inn1 and Cyk3 appear to cooperate in activating Chs2 for PS formation, which allows coordination of AMR contraction with ingression of the cleavage furrow.

post to: CiteULike

Loss of spindle assembly checkpoint-mediated inhibition of Cdc20 promotes tumorigenesis in mice.

Publication Date: 2009 Jun 15 PMID: 19528295
Authors: Li, M. - Fang, X. - Wei, Z. - York, J. P. - Zhang, P.
Journal: J Cell Biol

Genomic instability is a hallmark of human cancers. Spindle assembly checkpoint (SAC) is a critical cellular mechanism that prevents chromosome missegregation and therefore aneuploidy by blocking premature separation of sister chromatids. Thus, SAC, much like the DNA damage checkpoint, is essential for genome stability. In this study, we report the generation and analysis of mice carrying a Cdc20 allele in which three residues critical for the interaction with Mad2 were mutated to alanine. The mutant Cdc20 protein (AAA-Cdc20) is no longer inhibited by Mad2 in response to SAC activation, leading to the dysfunction of SAC and aneuploidy. The dysfunction could not be rescued by the additional expression of another Cdc20 inhibitor, BubR1. Furthermore, we found that Cdc20(AAA/AAA) mice died at late gestation, but Cdc20(+/AAA) mice were viable. Importantly, Cdc20(+/AAA) mice developed spontaneous tumors at highly accelerated rates, indicating that the SAC-mediated inhibition of Cdc20 is an important tumor-suppressing mechanism.

post to: CiteULike

Dynamic instability of microtubules requires dynamin 2 and is impaired in a Charcot-Marie-Tooth mutant.

Publication Date: 2009 Jun 15 PMID: 19528294
Authors: Tanabe, K. - Takei, K.
Journal: J Cell Biol

Dynamin is a fission protein that participates in endocytic vesicle formation. Although dynamin was originally identified as a microtubule-binding protein, the physiological relevance of this function was unclear. Recently, mutations in the ubiquitously expressed dynamin 2 (dyn2) protein were found in patients with Charcot-Marie-Tooth (CMT) disease, which is an inherited peripheral neuropathy. In this study, we show that one of these mutations, 551Delta3, induces prominent decoration of microtubules with the mutant dyn2. Dyn2 was required for proper dynamic instability of microtubules, and this was impaired in cells expressing the 551Delta3 mutant, which showed a remarkable increase in microtubule acetylation, a marker of stable microtubules. Depletion of endogenous dyn2 with a small interfering RNA also resulted in the accumulation of stable microtubules. Furthermore, the formation of mature Golgi complexes, which depends on microtubule-dependent membrane transport, was impaired in both dyn2 knockdown cells and cells expressing the 551Delta3 mutant. Collectively, our results suggest that dyn2 regulates dynamic instability of microtubules, which is essential for organelle motility, and that this function may be impaired in CMT disease.

post to: CiteULike

The role of aneuploidy in promoting and suppressing tumors.

Publication Date: 2009 Jun 15 PMID: 19528293
Authors: Weaver, B. A. - Cleveland, D. W.
Journal: J Cell Biol

Impaired mitotic checkpoint signaling can both promote and suppress tumors. The mitotic checkpoint targets Cdc20, the specificity factor of the ubiquitin ligase that promotes anaphase by targeting cyclin B and securin for destruction. In this issue, Li et al. (2009. J. Cell Biol. doi:10.1083/jcb.200904020) use gene replacement to produce mice expressing a Cdc20 mutant that cannot be inhibited by the mitotic checkpoint. In addition to the expected aneuploidy, these animals have a high tumor incidence that is likely caused by persistent aneuploidy coupled with nonmitotic functions of mutant Cdc20.

post to: CiteULike

Lucas Pelkmans: taking it from the top. Interview by Ben Short.

Publication Date: 2009 Jun 15 PMID: 19528292
Authors: Pelkmans, L.
Journal: J Cell Biol

post to: CiteULike

Distinct functions for Rho1 in maintaining adherens junctions and apical tension in remodeling epithelia.

Publication Date: 2009 Jun 15 PMID: 19506041
Authors: Warner, S. J. - Longmore, G. D.
Journal: J Cell Biol

Maintenance and remodeling of adherens junctions (AJs) and cell shape in epithelia are necessary for the development of functional epithelia and are commonly altered during cancer progression/metastasis. Although formation of nascent AJs has received much attention, whether shared mechanisms are responsible for the maintenance and remodeling of AJs in dynamic epithelia, particularly in vivo, is not clear. Using clonal analysis in the postmitotic Drosophila melanogaster pupal eye epithelium, we demonstrate that Rho1 is required to maintain AJ integrity independent of its role in sustaining apical cell tension. Rho1 depletion in a remodeling postmitotic epithelium disrupts AJs but only when depleted in adjacent cells. Surprisingly, neither of the Rho effectors, Rok or Dia, is necessary downstream of Rho1 to maintain AJs; instead, Rho1 maintains AJs by inhibiting Drosophila epithelial cadherin endocytosis in a Cdc42/Par6-dependent manner. In contrast, depletion of Rho1 in single cells decreases apical tension, and Rok and myosin are necessary, while Dia function also contributes, downstream of Rho1 to sustain apical cell tension.

post to: CiteULike

Regulators of yeast endocytosis identified by systematic quantitative analysis.

Publication Date: 2009 Jun 15 PMID: 19506040
Authors: Burston, H. E. - Maldonado-Baez, L. - Davey, M. - Montpetit, B. - Schluter, C. - Wendland, B. - Conibear, E.
Journal: J Cell Biol

Endocytosis of receptors at the plasma membrane is controlled by a complex mechanism that includes clathrin, adaptors, and actin regulators. Many of these proteins are conserved in yeast yet lack observable mutant phenotypes, which suggests that yeast endocytosis may be subject to different regulatory mechanisms. Here, we have systematically defined genes required for internalization using a quantitative genome-wide screen that monitors localization of the yeast vesicle-associated membrane protein (VAMP)/synaptobrevin homologue Snc1. Genetic interaction mapping was used to place these genes into functional modules containing known and novel endocytic regulators, and cargo selectivity was evaluated by an array-based comparative analysis. We demonstrate that clathrin and the yeast AP180 clathrin adaptor proteins have a cargo-specific role in Snc1 internalization. We additionally identify low dye binding 17 (LDB17) as a novel conserved component of the endocytic machinery. Ldb17 is recruited to cortical actin patches before actin polymerization and regulates normal coat dynamics and actin assembly. Our findings highlight the conserved machinery and reveal novel mechanisms that underlie endocytic internalization.

post to: CiteULike

Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo.

Publication Date: 2009 Jun 15 PMID: 19506039
Authors: Burgess, R. C. - Lisby, M. - Altmannova, V. - Krejci, L. - Sung, P. - Rothstein, R.
Journal: J Cell Biol

Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 "anti-recombinase" restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we cytologically characterize Srs2 function in vivo and describe a novel mechanism for regulating the initiation of HR. We find that Srs2 is recruited separately to replication and repair centers and identify the genetic requirements for recruitment. In the absence of Srs2 activity, Rad51 foci accumulate, and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2 removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR.

post to: CiteULike

Ups1p and Ups2p antagonistically regulate cardiolipin metabolism in mitochondria.

Publication Date: 2009 Jun 15 PMID: 19506038
Authors: Tamura, Y. - Endo, T. - Iijima, M. - Sesaki, H.
Journal: J Cell Biol

Cardiolipin, a unique phospholipid composed of four fatty acid chains, is located mainly in the mitochondrial inner membrane (IM). Cardiolipin is required for the integrity of several protein complexes in the IM, including the TIM23 translocase, a dynamic complex which mediates protein import into the mitochondria through interactions with the import motor presequence translocase-associated motor (PAM). In this study, we report that two homologous intermembrane space proteins, Ups1p and Ups2p, control cardiolipin metabolism and affect the assembly state of TIM23 and its association with PAM in an opposing manner. In ups1Delta mitochondria, cardiolipin levels were decreased, and the TIM23 translocase showed altered conformation and decreased association with PAM, leading to defects in mitochondrial protein import. Strikingly, loss of Ups2p restored normal cardiolipin levels and rescued TIM23 defects in ups1Delta mitochondria. Furthermore, we observed synthetic growth defects in ups mutants in combination with loss of Pam17p, which controls the integrity of PAM. Our findings provide a novel molecular mechanism for the regulation of cardiolipin metabolism.

post to: CiteULike

Sphingomyelin synthase-related protein SMSr controls ceramide homeostasis in the ER.

Publication Date: 2009 Jun 15 PMID: 19506037
Authors: Vacaru, A. M. - Tafesse, F. G. - Ternes, P. - Kondylis, V. - Hermansson, M. - Brouwers, J. F. - Somerharju, P. - Rabouille, C. - Holthuis, J. C.
Journal: J Cell Biol

Ceramides are central intermediates of sphingolipid metabolism with critical functions in cell organization and survival. They are synthesized on the cytosolic surface of the endoplasmic reticulum (ER) and transported by ceramide transfer protein to the Golgi for conversion to sphingomyelin (SM) by SM synthase SMS1. In this study, we report the identification of an SMS1-related (SMSr) enzyme, which catalyses the synthesis of the SM analogue ceramide phosphoethanolamine (CPE) in the ER lumen. Strikingly, SMSr produces only trace amounts of CPE, i.e., 300-fold less than SMS1-derived SM. Nevertheless, blocking its catalytic activity causes a substantial rise in ER ceramide levels and a structural collapse of the early secretory pathway. We find that the latter phenotype is not caused by depletion of CPE but rather a consequence of ceramide accumulation in the ER. Our results establish SMSr as a key regulator of ceramide homeostasis that seems to operate as a sensor rather than a converter of ceramides in the ER.

post to: CiteULike

During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation.

Publication Date: 2009 Jun 15 PMID: 19506036
Authors: Cohen, S. - Brault, J. J. - Gygi, S. P. - Glass, D. J. - Valenzuela, D. M. - Gartner, C. - Latres, E. - Goldberg, A. L.
Journal: J Cell Biol

Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1.

post to: CiteULike

Mammalian Fat and Dachsous cadherins regulate apical membrane organization in the embryonic cerebral cortex.

Publication Date: 2009 Jun 15 PMID: 19506035
Authors: Ishiuchi, T. - Misaki, K. - Yonemura, S. - Takeichi, M. - Tanoue, T.
Journal: J Cell Biol

Compartmentalization of the plasma membrane in a cell is fundamental for its proper functions. In this study, we present evidence that mammalian Fat4 and Dachsous1 cadherins regulate the apical plasma membrane organization in the embryonic cerebral cortex. In neural progenitor cells of the cortex, Fat4 and Dachsous1 were concentrated together in a cell-cell contact area positioned more apically than the adherens junction (AJ). These molecules interacted in a heterophilic fashion, affecting their respective protein levels. We further found that Fat4 associated and colocalized with the Pals1 complex. Ultrastructurally, the apical junctions of the progenitor cells comprised the AJ and a stretch of plasma membrane apposition extending apically from the AJ, which positionally corresponded to the Fat4-Dachsous1-positive zone. Depletion of Fat4 or Pals1 abolished this membrane apposition. These results highlight the importance of the Fat4-Dachsous1-Pals1 complex in organizing the apical membrane architecture of neural progenitor cells.

post to: CiteULike

Acetylation and activation of STAT3 mediated by nuclear translocation of CD44.

Publication Date: 2009 Jun 15 PMID: 19506034
Authors: Lee, J. L. - Wang, M. J. - Chen, J. Y.
Journal: J Cell Biol

Expression of the type I transmembrane glycoprotein CD44 has recently been recognized as a signature for cancer stem cells. In this study, we demonstrate that CD44, once engaged, is internalized and translocated to the nucleus, where it binds to various promoters, including that of cyclin D1, leading to cell fate change through transcriptional reprogramming. In regulating cyclin D1 expression, the internalized CD44 forms a complex with STAT3 and p300 (acetyltransferase), eliciting STAT3 acetylation at lysine 685 and dimer formation in a cytokine- and growth factor-independent manner. A bipartite nuclear localization signal (NLS) was mapped to the cytoplasmic tail of CD44, which mediates its nuclear translocation. Expression of CD44(NLS) mutant sequesters STAT3 in cytosol. In the nucleus, the acetylated STAT3 dimer remains associated with CD44 and binds to the cyclin D1 promoter, leading to increased cyclin D1 expression and cell proliferation. This study describes a novel function for CD44 in transcriptional modulation through nuclear translocation of the internalized CD44 and complex formation with transcription factors.

post to: CiteULike