Prohibitins constitute an evolutionarily conserved and ubiquitously expressed family of membrane proteins that are essential for cell proliferation and development in higher eukaryotes. Roles for prohibitins in cell signaling at the plasma membrane and in transcriptional regulation in the nucleus have been proposed, but pleiotropic defects associated with the loss of prohibitin genes can be largely attributed to a dysfunction of mitochondria. Two closely related proteins, prohibitin-1 (PHB1) and prohibitin-2 (PHB2), form large, multimeric ring complexes in the inner membrane of mitochondria. The absence of prohibitins leads to an increased generation of reactive oxygen species, disorganized mitochondrial nucleoids, abnormal cristae morphology and an increased sensitivity towards stimuli-elicited apoptosis. It has been found that the processing of the dynamin-like GTPase OPA1, which regulates mitochondrial fusion and cristae morphogenesis, is a key process regulated by prohibitins. Furthermore, genetic analyses in yeast have revealed an intimate functional link between prohibitin complexes and the membrane phospholipids cardiolipin and phosphatidylethanolamine. In light of these findings, it is emerging that prohibitin complexes can function as protein and lipid scaffolds that ensure the integrity and functionality of the mitochondrial inner membrane.

Faithful segregation of genetic material during cell division requires the dynamic but robust attachment of chromosomes to spindle microtubules during all stages of mitosis. This regulated attachment occurs at kinetochores, which are complex protein organelles that are essential for cell survival and genome integrity. In budding yeast, in which a single microtubule attaches per kinetochore, a heterodecamer known as the Dam1 complex (or DASH complex) is required for proper chromosome segregation. Recent years have seen a burst of structural and biophysical data concerning this interesting complex, which has caught the attention of the mitosis research field. In vitro, the Dam1 complex interacts directly with tubulin and self-assembles into ring structures around the microtubule surface. The ring is capable of tracking with depolymerizing ends, and a model has been proposed whereby the circular geometry of the oligomeric Dam1 complex allows it to couple the depolymerization of microtubules to processive chromosome movement in the absence of any additional energy source. Although it is attractive and simple, several important aspects of this model remain controversial. Additionally, the generality of the Dam1 mechanism has been questioned owing to the fact that there are no obvious Dam1 homologs beyond fungi. In this Commentary, we discuss recent structure-function studies of this intriguing complex.

It has long been known that phosphoinositides are present in cellular membranes, but only in the past four decades has our understanding of their importance for proper cell function advanced significantly. Key to determining the biological roles of phosphoinositides is understanding the enzymes involved in their metabolism. Although many such enzymes have now been identified, there is still much to learn about their cellular functions. Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) are a group of kinases that catalyse the production of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P₂]. As well as being a substrate for the enzymes phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K), PtdIns(4,5)P₂ acts as a second messenger in its own right, influencing a variety of cellular processes. In this Commentary, we review how PIP5Ks are modulated to achieve regulated PtdIns(4,5)P₂ production, and discuss the role of these proteins in different cellular processes.

Absence or mutation of keratins 8 (K8) or 18 (K18) cause predisposition to liver injury and apoptosis. We assessed the mechanisms of hepatocyte keratin-mediated cytoprotection by comparing the protein expression profiles of livers from wild-type and K8-null mice using two-dimensional differential-in-gel-electrophoresis (2D-DIGE) and mass spectrometry. Prominent among the alterations were those of mitochondrial proteins, which were confirmed using 2D-DIGE of purified mitochondria. Ultrastructural analysis showed that mitochondria of livers that lack or have disrupted keratins are significantly smaller than mitochondria of wild-type livers. Immunofluorescence staining showed irregular distribution of mitochondria in keratin-absent or keratin-mutant livers. K8-null livers have decreased ATP content; and K8-null mitochondria have less cytochrome c, increased release of cytochrome c after exposure to Ca²⁺ and oxidative stimulation, and a higher sensitivity to Ca²⁺-induced permeability transition. Therefore, keratins play a direct or indirect role in regulating the shape and function of mitochondria. The effects of keratin mutation on mitochondria are likely to contribute to hepatocyte predisposition to apoptosis and oxidative injury, and to play a pathogenic role in keratin-mutation-related human liver disease.

Type I myosins are monomeric motors involved in a range of motile and sensory activities in different cell types. In simple unicellular eukaryotes, motor activity of class I myosins is regulated by phosphorylation of a conserved `TEDS site' residue within the motor domain. The mechanism by which this phosphorylation event affects the cellular function of each myosin I remains unclear. The fission yeast myosin I, Myo1, activates Arp2/3-dependent polymerisation of cortical actin patches and also regulates endocytosis. Using mutants and Myo1-specific antibodies, we show that the phosphorylation of the Myo1 TEDS site (serine 361) plays a crucial role in regulating this protein's dynamic localisation and cellular function. We conclude that although phosphorylation of serine 361 does not affect the ability of this motor protein to promote actin polymerisation, it is required for Myo1 to recruit to sites of endocytosis and function during this process.

Coordination between microtubule and actin cytoskeletons plays a crucial role during the establishment of cell polarity. In fission yeast, the microtubule cytoskeleton regulates the distribution of actin assembly at the new growing end during the monopolar-to-bipolar growth transition. Here, we describe a novel mechanism in which a myosin V modulates the spatial coordination of proteolysis and microtubule dynamics. In cells lacking a functional copy of the class V myosin, Myo52, the plus ends of microtubules fail to undergo catastrophe on contacting the cell end and continue to grow, curling around the end of the cell. We show that this actin-associated motor regulates the efficient ubiquitin-dependent proteolysis of the Schizosaccharomyces pombe CLIP-170 homologue, Tip1. Myo52 facilitates microtubule catastrophe by enhancing Tip1 removal from the plus end of growing microtubules at the cell tips. There, Myo52 and the ubiquitin receptor, Dph1, work in concert to target Tip1 for degradation.

Podosomes, adhesion structures capable of matrix degradation, have been linked with the ability of cells to perform chemotaxis and invade tissues. Wiskott-Aldrich Syndrome protein (WASp), an effector of the RhoGTPase Cdc42 and a Src family kinase substrate, regulates macrophage podosome formation. In this study, we demonstrate that WASp is active in podosomes by using TIRF-FRET microscopy. Pharmacological and RNA interference approaches suggested that continuous WASp activity is required for podosome formation and function. Rescue experiments using point mutations demonstrate an absolute requirement for Cdc42 binding to WASp in podosome formation. Although tyrosine phosphorylation was not absolutely required for podosome formation, phosphorylation did regulate the rate of podosome nucleation and actin filament stability. Importantly, WASp tyrosine phosphorylation does not alter WASp activation, instead phosphorylation appears to be important for the restriction of WASp activity to podosomes. In addition, the matrix-degrading ability of cells requires WASp phosphorylation. Chemotactic responses to CSF-1 were also attenuated in the absence of endogenous WASp, which could not be rescued with either tyrosine mutation. These results suggest a more complex role for tyrosine phosphorylation than simply in the regulation of WASp activity, and suggest a link between podosome dynamics and macrophage migration.

Gap junctions are dynamic plasma membrane domains, and their protein constituents, the connexins, have a high turnover rate in most tissue types. However, the molecular mechanisms involved in degradation of gap junctions have remained largely unknown. Here, we show that ubiquitin is strongly relocalized to connexin-43 (Cx43; also known as Gja1) gap junction plaques in response to activation of protein kinase C. Cx43 remained ubiquitylated during its transition to a Triton X-100-soluble state and along its trafficking to early endosomes. Following internalization, Cx43 partly colocalized with the ubiquitin-binding proteins Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate; also known as Hgs) and Tsg101 (tumor susceptibility gene 101). Depletion of Hrs or Tsg101 by small interfering RNA abrogated trafficking of Cx43 from early endosomes to lysosomes. Under these conditions, Cx43 was able to undergo dephosphorylation and deubiquitylation, locate to the plasma membrane and form functional gap junctions. Simultaneous depletion of Hrs and Tsg101 caused accumulation of a phosphorylated and ubiquitylated subpopulation of Cx43 in early endosomes and in hybrid organelles between partly degraded annular gap junctions and endosomes. Collectively, these data reveal a central role of early endosomes in sorting of ubiquitylated Cx43, and identify Hrs and Tsg101 as crucial regulators of trafficking of Cx43 to lysosomes.

Mouse sperm-egg binding requires a multiplicity of receptor-ligand interactions, including an oviduct-derived, high molecular weight, wheat germ agglutinin (WGA)-binding glycoprotein that associates with the egg coat at ovulation. Herein, we report the purification and identification of this sperm-binding ligand. WGA-binding, high molecular weight glycoproteins isolated from hormonally primed mouse oviduct lysates competitively inhibit sperm-egg binding in vitro. Within this heterogeneous glycoprotein preparation, a distinct 220 kDa protein selectively binds to sperm surfaces, and was identified by sequence analysis as oviduct-specific glycoprotein (OGP). The sperm-binding activity of OGP was confirmed by the loss of sperm-binding following immunodepletion of OGP from oviduct lysates, and by the ability of both immunoprecipitated OGP and natively purified OGP to competitively inhibit sperm-egg binding. As expected, OGP is expressed by the secretory cells of the fimbriae and infundibulum; however, in contrast to previous reports, OGP is also associated with both the zona pellucida and the perivitelline space of mouse oocytes. Western blot analysis and lectin affinity chromatography demonstrate that whereas the bulk of OGP remains soluble in the ampullar fluid, distinct glycoforms associate with the cumulus matrix, zona pellucida and perivitelline space. The sperm-binding activity of OGP is carbohydrate-dependent and restricted to a relatively minor peanut agglutinin (PNA)-binding glycoform that preferentially associates with the sperm surface, zona pellucida and perivitelline space, relative to other more abundant glycoforms. Finally, pretreatment of two-cell embryos, which do not normally bind sperm, with PNA-binding OGP stimulates sperm binding.

Cyclic AMP has a crucial role during the entire developmental program of the social amoebae Dictyostelium, acting both as an intracellular second messenger and, when secreted, as a directional cue that is relayed to neighboring cells during chemotaxis. Although significant knowledge about cAMP production in chemotaxing cells has been derived from studies performed on cell populations, cAMP dynamics at the single cell level have not been investigated. To examine this, we used a FRET-based cAMP sensor that possesses high cAMP sensitivity and great temporal resolution. We show the transient profile of cAMP accumulation in live Dictyostelium cells and establish that chemoattractants control intracellular cAMP dynamics by regulating synthesis via the adenylyl cyclase ACA. aca^– cells show no significant change in FRET response following chemoattractant addition. Furthermore, cells lacking ACB, the other adenylyl cyclase expressed in chemotaxing cells, behave similarly to wild-type cells. We also establish that the RegA is the major phosphodiesterase that degrades intracellular cAMP in chemotaxis-competent cells. Interestingly, we failed to measure intracellular cAMP compartmentalization in actively chemotaxing cells. We conclude that cytosolic cAMP, which is destined to activate PKA, is regulated by ACA and RegA and does not compartmentalize during chemotaxis.

Stimulation of Na⁺/K⁺-ATPase activity in alveolar epithelial cells by cAMP involves its recruitment from intracellular compartments to the plasma membrane. Here, we studied the role of the actin molecular motor myosin-V in this process. We provide evidence that, in alveolar epithelial cells, cAMP promotes Na⁺/K⁺-ATPase recruitment to the plasma membrane by increasing the average speed of Na⁺/K⁺-ATPase-containing vesicles moving to the cell periphery. We found that three isoforms of myosin-V are expressed in alveolar epithelial cells; however, only myosin-Va and Vc colocalized with the Na⁺/K⁺-ATPase in intracellular membrane fractions. Overexpression of dominant-negative myosin-Va or knockdown with specific shRNA increased the average speed and distance traveled by the Na⁺/K⁺-ATPase-containing vesicles, as well as the Na⁺/K⁺-ATPase activity and protein abundance at the plasma membrane to similar levels as those observed with cAMP stimulation. These data show that myosin-Va has a role in restraining Na⁺/K⁺-ATPase-containing vesicles within intracellular pools and that this restrain is released after stimulation by cAMP allowing the recruitment of the Na⁺/K⁺-ATPase to the plasma membrane and thus increased activity.

During lymphatic development, Prox1 plays central roles in the differentiation of blood vascular endothelial cells (BECs) into lymphatic endothelial cells (LECs), and subsequently in the maturation and maintenance of lymphatic vessels. However, the molecular mechanisms by which Prox1 elicits these functions remain to be elucidated. Here, we identified FoxC2 and angiopoietin-2 (Ang2), which play important roles in the maturation of lymphatic vessels, as novel targets of Prox1 in mouse embryonic-stem-cell-derived endothelial cells (MESECs). Furthermore, we found that expression of HoxD8 was significantly induced by Prox1 in MESECs, a finding confirmed in human umbilical vein endothelial cells (HUVECs) and human dermal LECs (HDLECs). In mouse embryos, HoxD8 expression was significantly higher in LECs than in BECs. In a model of inflammatory lymphangiogenesis, diameters of lymphatic vessels of the diaphragm were increased by adenovirally transduced HoxD8. We also found that HoxD8 induces Ang2 expression in HDLECs and HUVECs. Moreover, we found that HoxD8 induces Prox1 expression in HUVECs and that knockdown of HoxD8 reduces this expression in HDLECs, suggesting that Prox1 expression in LECs is maintained by HoxD8. These findings indicate that transcriptional networks of Prox1 and HoxD8 play important roles in the maturation and maintenance of lymphatic vessels.

Programmed cell death is induced by the activation of a subset of intracellular proteins in response to specific extra- and intracellular signals. In the yeast Saccharomyces cerevisiae, Nma111p functions as a nuclear serine protease that is necessary for apoptosis under cellular stress conditions, such as elevated temperature or treatment of cells with hydrogen peroxide to induce cell death. We have examined the role of nuclear protein import in the function of Nma111p in apoptosis. Nma111p contains two small clusters of basic residues towards its N-terminus, both of which are necessary for efficient translocation into the nucleus. Nma111p does not shuttle between the nucleus and cytoplasm during either normal growth conditions or under environmental stresses that induce apoptosis. The N-terminal half of Nma111p is sufficient to provide the apoptosis-inducing activity of the protein, and the nuclear-localisation signal (NLS) sequences and catalytic serine 235 are both necessary for this function. We provide compelling evidence that intranuclear Nma111p activity is necessary for apoptosis in yeast.

Missense mutations in human PLP1, the gene encoding myelin proteolipid protein (PLP), cause dysmyelinating Pelizaeus-Merzbacher disease of varying severity. Although disease pathology has been linked to retention of misfolded PLP in the endoplasmic reticulum (ER) and induction of the unfolded protein response (UPR), the molecular mechanisms that govern phenotypic heterogeneity remain poorly understood. To address this issue, we examined the cellular response to missense mutants of PLP that are associated with distinct disease phenotypes. We found that the mild-disease-associated mutants, W162L and G245A, were cleared from the ER comparatively quickly via proteasomal degradation and/or ER exit. By contrast, the more `aggressive' A242V mutant, which causes severe disease, was significantly more stable, accumulated at the ER and resulted in a specific activation of the UPR. On the basis of these findings, we propose that the rate at which mutant PLP proteins are cleared from the ER modulates disease severity by determining the extent to which the UPR is activated.

Proteins of the Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family link signal transduction pathways to actin cytoskeleton dynamics. VASP is substrate of cAMP-dependent, cGMP-dependent and AMP-activated protein kinases that primarily phosphorylate the sites S157, S239 and T278, respectively. Here, we systematically analyzed functions of VASP phosphorylation patterns for actin assembly and subcellular targeting in vivo and compared the phosphorylation effects of Ena/VASP family members. Methods used were the reconstitution of VASP-null cells with `locked' phosphomimetic VASP mutants, actin polymerization of VASP mutants in vitro and in living cells, site-specific kinase-mediated VASP phosphorylation, and analysis of the endogenous protein with phosphorylation-status-specific antibodies. Phosphorylation at S157 influenced VASP localization, but had a minor impact on F-actin assembly. Phosphorylation of the S157-equivalent site in the Ena/VASP family members Mena and EVL had no effect on the ratio of cellular F-actin to G-actin. By contrast, VASP phosphorylation at S239 (and the equivalent site in Mena) or T278 impaired VASP-driven actin filament formation. The data show that VASP functions are precisely regulated by differential phosphorylation and provide new insights into cytoskeletal control by serine/threonine kinase-dependent signaling pathways.

It remains unclear how GPI-anchored proteins (GPIAPs), which lack cytoplasmic domains, transduce signals triggered by specific ligation. Such signal transduction has been speculated to require the ligated GPIAP to associate with membrane-spanning proteins that communicate with obligate cytoplasmic proteins. Transient anchorage of crosslinked proteins on the cell surface was previously characterized by single-particle tracking, and temporary association with the actin cytoskeleton was hypothesized to cause regulated anchorage. GPIAPs, such as Thy-1, require clustering, cholesterol and Src-family kinase (SFK) activity to become transiently anchored. By contrast, a transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), which has a PDZ-binding motif in its cytoplasmic C-terminus that binds the ERM adaptor EBP50, exhibits anchorage that strictly requires EBP50 but has little dependence on cholesterol or SFK. We hypothesized that a transmembrane protein would be required to mediate the linkage between Thy-1 and the cytoskeleton. Here, we present evidence, obtained by shRNA knockdown, that the transmembrane protein Csk-binding protein (CBP) plays an obligatory role in the transient anchorage of Thy1. Furthermore, either a dominant-negative form of CBP that did not bind EBP50 or a dominant-negative EBP50 drastically reduced transient anchorage of Thy-1, indicating the involvement of this adaptor. Finally, we speculate on the role of phosphorylation in the regulation of transient anchorage.

Stress granules (SGs) and P-bodies (PBs) are related cytoplasmic structures harboring silenced mRNAs. SGs assemble transiently upon cellular stress, whereas PBs are constitutive and are further induced by stress. Both foci are highly dynamic, with messenger ribonucleoproteins (mRNPs) and proteins rapidly shuttling in and out. Here, we show that impairment of retrograde transport by knockdown of mammalian dynein heavy chain 1 (DHC1) or bicaudal D1 (BicD1) inhibits SG formation and PB growth upon stress, without affecting protein-synthesis blockage. Conversely, impairment of anterograde transport by knockdown of kinesin-1 heavy chain (KIF5B) or kinesin light chain 1 (KLC1) delayed SG dissolution. Strikingly, SG dissolution is not required to restore translation. Simultaneous knockdown of dynein and kinesin reverted the effect of single knockdowns on both SGs and PBs, suggesting that a balance between opposing movements driven by these molecular motors governs foci formation and dissolution. Finally, we found that regulation of SG dynamics by dynein and kinesin is conserved in Drosophila.

The Arp2/3 complex is important for morphogenesis in various developmental systems, but specific in vivo roles for this complex in cells that move during morphogenesis are not well understood. We have examined cellular roles for Arp2/3 in the Caenorhabditis elegans embryo. In C. elegans, the first morphogenetic movement, gastrulation, is initiated by the internalization of two endodermal precursor cells. These cells undergo a myosin-dependent apical constriction, pulling a ring of six neighboring cells into a gap left behind on the ventral surface of the embryo. In agreement with a previous report, we found that in Arp2/3-depleted C. elegans embryos, membrane blebs form and the endodermal precursor cells fail to fully internalize. We show that these cells are normal with respect to several key requirements for gastrulation: cell cycle timing, cell fate, apicobasal cell polarity and apical accumulation and activation of myosin-II. To further understand the function of Arp2/3 in gastrulation, we examined F-actin dynamics in wild-type embryos. We found that three of the six neighboring cells extend short, dynamic F-actin-rich processes at their apical borders with the internalizing cells. These processes failed to form in embryos that were depleted of Arp2/3 or the apical protein PAR-3. Our results identify an in vivo role for Arp2/3 in the formation of subcellular structures during morphogenesis. The results also suggest a new layer to the model of C. elegans gastrulation: in addition to apical constriction, internalization of the endoderm might involve dynamic Arp2/3-dependent F-actin-rich extensions on one side of a ring of cells.

Autosomal dominant neurohypophyseal diabetes insipidus results from mutations in the precursor protein of the antidiuretic hormone arginine vasopressin. Mutant prohormone is retained in the endoplasmic reticulum of vasopressinergic neurons and causes their progressive degeneration by an unknown mechanism. Here, we show that several dominant pro-vasopressin mutants form disulfide-linked homo-oligomers and develop large aggregations visible by immunofluorescence and immunogold electron microscopy, both in a fibroblast and a neuronal cell line. Double-labeling showed the pro-vasopressin aggregates to colocalize with the chaperone calreticulin, indicating that they originated from the endoplasmic reticulum. The aggregates revealed a remarkable fibrillar substructure. Bacterially expressed and purified mutant pro-vasopressin spontaneously formed fibrils under oxidizing conditions. Mutagenesis experiments showed that the presence of cysteines, but no specific single cysteine, is essential for disulfide oligomerization and aggregation in vivo. Our findings assign autosomal dominant diabetes insipidus to the group of neurodegenerative diseases associated with the formation of fibrillar protein aggregates.

The senile plaques found in the brains of patients with Alzheimer's disease are mainly due to the accumulation of amyloid β-peptides (Aβ) that are liberated by -secretase, a high molecular weight complex including presenilins, PEN-2, APH-1 and nicastrin. The depletion of each of these proteins disrupts the complex assembly into a functional protease. Here, we describe another level of regulation of this multimeric protease. The depletion of both presenilins drastically reduces Pen2 mRNA levels and its promoter transactivation. Furthermore, overexpression of presenilin-1 lowers Pen2 promoter transactivation, a phenotype abolished by a double mutation known to prevent presenilin-dependent -secretase activity. PEN-2 expression is decreased by depletion of β-amyloid precursor protein (APP) and increased by the APP intracellular domain (AICD). We show that AICD and APP complement for Pen2 mRNA levels in APP/APLP1-2 knockout fibroblasts. Interestingly, overexpression of presenilin-2 greatly increases Pen2 promoter transactivation. The opposite effect triggered by both presenilins was reminiscent of our previous study, which showed that these two proteins elicit antagonistic effects on p53. Therefore, we examined the contribution of p53 on Pen2 transcription. Pen2 promoter transactivation, and Pen2 mRNA and protein levels were drastically reduced in p53^–/– fibroblasts. Furthermore, PEN-2 expression could be rescued by p53 complementation in p53- and APP-deficient cells. Interestingly, PEN-2 expression was also reduced in p53-deficient mouse brain. Overall, our study describes a p53-dependent regulation of PEN-2 expression by other members of the -secretase complex, namely presenilins.