Throughout normal development, and in aberrant conditions such as cancer, cells divide by a process called cytokinesis. Most textbooks suggest that animal cells execute cytokinesis using an actomyosin-containing contractile ring, whereas plant cells generate a new cell wall by the assembly of a novel membrane compartment using vesicle-trafficking machinery in an apparently distinct manner. Recent studies have shown that cytokinesis in animal and plant cells may not be as distinct as these models imply – both have an absolute requirement for vesicle traffic. Moreover, some of the key molecular components of cytokinesis have been identified, many of which are proteins that function to control membrane traffic. Here, we review recent advances in this area.
]]>The GTPase Ran has a key role in nuclear import and export, mitotic spindle assembly and nuclear envelope formation. The cycling of Ran between its GTP- and GDP-bound forms is catalyzed by the chromatin-bound guanine nucleotide exchange factor RCC1 and the cytoplasmic Ran GTPase-activating protein RanGAP. The result is an intracellular concentration gradient of RanGTP that equips eukaryotic cells with a `genome-positioning system' (GPS). The binding of RanGTP to nuclear transport receptors (NTRs) of the importin β superfamily mediates the effects of the gradient and generates further downstream gradients, which have been elucidated by fluorescence resonance energy transfer (FRET) imaging and computational modeling. The Ran-dependent GPS spatially directs many functions required for genome segregation by the mitotic spindle during mitosis. Through exportin 1, RanGTP recruits essential centrosome and kinetochore components, whereas the RanGTP-induced release of spindle assembly factors (SAFs) from importins activates SAFs to nucleate, bind and organize nascent...]]>
Two lipids (palmitate and palmitoleic acid) are appended onto Wnt proteins. It has been suggested that palmitate is required for signalling, whereas palmitoleic acid is necessary for progression through the secretory pathway. By mutating the relevant amino acids, we have investigated how these adducts contribute to the secretion and signalling activity of Wingless, the main Drosophila member of the Wnt family. Analysis of Wingless with a Cysteine 93 to Alanine mutation ([C93A]Wingless) shows that palmitoylation is essential for signalling activity in vivo (as well as in cultured cells). Moreover, without palmitate, Wingless fails to reach the surface of imaginal disc cells and, as electron microscopy (EM) analysis suggests, appears to accumulate in the endoplasmic reticulum (ER). Artificial targeting of palmitate-deficient Wingless to the plasma membrane does not rescue signalling activity. Therefore, palmitate at C93 has a dual role: in secretion and signalling. From our analysis of [S239A]Wingless, which lacks...]]>
Mutations in the gene encoding the basal lamina (BL) component laminin 2 (LAMA2) cause merosin-deficient congenital muscular dystrophy 1A (MDC1A), a complex disorder that includes hypomyelination and myodegeneration. In dystrophia muscularis (dy) mice bearing Lama2 mutations, myofibers and Schwann cells fail to assemble stable BLs, which are thought to be crucial for myofiber survival and Schwann cell differentiation. Here, we describe defects in a new allele of Lama2 in mice, nmf417, in which a point mutation substitutes Arg for Cys79 at a universally conserved CxxC motif in the laminin N-terminal (LN) domain; this domain mediates laminin-laminin interactions. nmf417 homozygosity caused progressive myodegeneration and severe peripheral amyelination in nerve roots, similar to previous Lama2 mutations, but without the pervasive BL thinning previously associated with the disorder. In direct contrast to the previously characterized dy and dy2J alleles, nmf417 homozygous myofibers frequently had thickened BLs. Severe amyelination in nmf417-mutant nerve roots...]]>
Primary spermatocyte nuclei of Drosophila melanogaster contain three prominent lampbrush-like loops. The development of these structures has been associated with the transcription of three fertility factors located on the Y chromosome, named kl-5, kl-3 and ks-1. These loci have huge physical dimensions and contain extremely long introns. In addition, kl-3 and kl-5 were shown to encode two putative dynein subunits required for the correct assembly of the sperm axoneme. Here, we show that both the kl-5 and kl-3 loops are intensely decorated by monoclonal antibodies recognizing triple-stranded nucleic acids, and that each loop presents a peculiar molecular organization of triplex structures. Moreover, immunostaining of Drosophila hydei primary spermatocytes revealed that also in this species – which diverged from D. melanogaster 58 million years ago – Y-loops are decorated by anti-triplex antibodies, strongly suggesting a conserved role of loop-associated triplexes. Finally, we showed that in D. melanogaster wild-type lines that...]]>
Notch signaling is required for multiple aspects of tissue and cell differentiation. In this study, we identified zinc finger protein 64 (Zfp64) as a novel coactivator of Notch1. Zfp64 is associated with the intracellular domain of Notch1, recruited to the promoters of the Notch target genes Hes1 and Hey1, and transactivates them. Zfp64 expression is under the control of Runx2, and is upregulated by direct transactivation of its promoter. Zfp64 suppresses the myogenic differentiation of C2C12 cells and promotes their osteoblastic differentiation. Our data demonstrate two functions of Zfp64: (1) it is a downstream target of Runx2 and, (2) its cognate protein acts as a coactivator of Notch1, which suggests that Zfp64 mediates mesenchymal cell differentiation by modulating Notch signaling.
]]>Chloroquine resistance in the malaria parasite Plasmodium falciparum has made malaria increasingly difficult to control. Chloroquine-resistant parasites accumulate less chloroquine than their chloroquine-sensitive counterparts; however, the mechanism underlying this remains unclear. The primary site of accumulation and antimalarial action of chloroquine is the internal acidic digestive vacuole of the parasite, the acidity of which is maintained by inwardly-directed H+ pumps, working against the (outward) leak of H+. In this study we have investigated the leak of H+ from the digestive vacuole of the parasite by monitoring the alkalinisation of the vacuole following inhibition of the H+-pumping V-type ATPase by concanamycin A. The rates of alkalinisation observed in three chloroquine-resistant strains were two- to fourfold higher than those measured in three chloroquine-sensitive strains. On addition of chloroquine there was a dramatic increase in the rate of alkalinisation in the chloroquine-resistant strains, whereas chloroquine caused the rate of alkalinisation to decrease...]]>
Yeast peroxisomes multiply by fission. Fission requires two dynamin-related proteins, Dnm1p and Vps1p. Using an in vivo fission assay, we show that Dnm1p-dependent peroxisome fission requires Fis1p, Caf4p and Mdv1p. Fluorescence microscopy of cells expressing GFP-tagged Caf4p and Mdv1p revealed that their association with peroxisomes relies on Fis1p. Vps1p-dependent peroxisome fission occurs independently of these factors. Vps1p contributes most to fission of peroxisomes when cells are grown on glucose. Overexpression of Dnm1p suppresses the fission defect as long as Fis1p and either Mdv1p or Caf4p are present. Conversely, overexpression of Dnm1p does not restore the vacuolar fusion defect of vps1 cells and Vps1p overexpression does not restore the mitochondrial fission defect of dnm1 cells. These data show that Vps1p and Dnm1p are part of independent fission machineries. Because the contribution of Dnm1p to peroxisome fission appears to be more pronounced in cells that proliferate peroxisomes in response to mitochondrial...]]>
Recent studies have shown that Src-family kinases (SFKs) play an important role in mediating integrin signalling, and the β3 subunit of IIbβ3 integrin has been shown to interact with multiple SFK members. Here, we analyzed the interactions and functional consequences of Fyn and Src binding to IIbβ3. Fyn associated with the β3 subunit in resting and thrombin-aggregated platelets, whereas interaction between Src and IIbβ3 was seen predominantly in resting but not in thrombin-aggregated platelets. We have also observed that Fyn but not Src localized to focal adhesions in CHO cells adherent to fibrinogen through IIbβ3. On the basis of these differences, we wanted to determine the sequence requirements for the interaction of Fyn and Src within the β3-cytoplasmic domain. Whereas Src association required the C-terminal region of β3, Fyn continued to interact with mutants that could no longer associate with Src and that contained as few as 13 membrane-proximal...]]>
Huntington disease (HD) is caused by a polyglutamine-expansion mutation in huntingtin (HTT) that makes the protein toxic and aggregate-prone. The subcellular localisation of huntingtin and many of its interactors suggest a role in endocytosis, and recently it has been shown that huntingtin interacts indirectly with the early endosomal protein Rab5 through HAP40. Here we show that Rab5 inhibition enhanced polyglutamine toxicity, whereas Rab5 overexpression attenuated toxicity in our cell and fly models of HD. We tried to identify a mechanism for the Rab5 effects in our HD model systems, and our data suggest that Rab5 acts at an early stage of autophagosome formation in a macromolecular complex that contains beclin 1 (BECN1) and Vps34. Interestingly chemical or genetic inhibition of endocytosis also impeded macroautophagy, and enhanced aggregation and toxicity of mutant huntingtin. However, in contrast to Rab5, inhibition of endocytosis by various means suppressed autophagosome-lysosome fusion (the final step...]]>
The plasminogen activation system regulates matrix remodeling through both proteolytic and non-proteolytic mechanisms. Studies were undertaken to determine the effects of the plasminogen activator inhibitor 1 (PAI1) on the assembly of the fibronectin matrix. The addition of PAI1 to MG-63 cells caused a 1.5- to threefold increase in the rate of fibronectin matrix assembly which was associated with an increase in β integrin activation. PAI1 treatment led to a marked decrease in focal contacts and stress fibers, whereas tensin-containing matrix contacts remained unaffected. The effects of PAI1 on matrix assembly were independent of both urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR), indicating that the stimulation of matrix assembly by PAI1 does not depend on its anti-proteolytic activity or on the association of uPAR with integrin receptors. Antagonists of the vβ5 integrin mimicked the effect of PAI1 on cell morphology and fibronectin matrix deposition, indicating that stimulation...]]>
SPARC is an evolutionarily conserved collagen-binding extracellular matrix (ECM) glycoprotein whose morphogenetic contribution(s) to embryonic development remain elusive despite decades of research. We have therefore used Drosophila genetics to gain insight into the role of SPARC during embryogenesis. In Drosophila embryos, high levels of SPARC and other basal lamina components (such as network-forming collagen IV, laminin and perlecan) are synthesized and secreted by haemocytes, and assembled into basal laminae. A SPARC mutant was generated by P-element mutagenesis that is embryonic lethal because of multiple developmental defects. Whereas no differences in collagen IV immunostaining were observed in haemocytes between wild-type and SPARC-mutant embryos, collagen IV was not visible in basal laminae of SPARC-mutant embryos. In addition, the laminin network of SPARC-mutant embryos appeared fragmented and discontinuous by late embryogenesis. Transgenic expression of SPARC protein by haemocytes in SPARC-mutant embryos restored collagen IV and laminin continuity in basal laminae. However, transgenic...]]>
Cells that are treated long-term with TNF or short-term with TGF together with cycloheximide (CHX) undergo apoptosis. Cell shrinkage and detachment during apoptosis is dependent on actomyosin contractility. Myosin II heavy chain (MHCII) isoforms have shared and distinct functions. Here, we investigated whether the involvement of MHCII isoforms A and B (MHCIIA and MHCIIB, respectively) in cell shrinkage and detachment differs during apoptosis. We show that TNF induces caspase-dependent MHCIIA degradation, whereas MHCIIB levels and association with the cytoskeleton remained virtually unchanged in TtT/GF cells and NIH 3T3 fibroblasts. MHCIIA proteolysis also occurred in fibroblasts that lack MHCIIB when treated with TNF and CHX together. The absence of MHCIIB did not affect cell death rate. However, MHCIIB–/– cells showed more resistance to TNF–induced actin disassembly, cell shrinkage and detachment than wild-type fibroblasts, indicating the participation of MHCIIB in these events. Moreover, inhibition of atypical PKC, which targets MHCIIB but...]]>
SUUR (Suppressor of Under-Replication) protein is responsible for late replication and, as a consequence, for DNA underreplication of intercalary and pericentric heterochromatin in Drosophila melanogaster polytene chromosomes. However, the mechanism by which SUUR slows down the replication process is not clear. To identify possible partners for SUUR we performed a yeast two-hybrid screen using full-length SUUR as bait. This identified HP1, the well-studied heterochromatin protein, as a strong SUUR interactor. Furthermore, we have determined that the central region of SUUR is necessary and sufficient for interaction with the C-terminal part of HP1, which contains the hinge and chromoshadow domains. In addition, recruitment of SUUR to ectopic HP1 sites on chromosomes provides evidence for their association in vivo. Indeed, we found that the distributions of SUUR and HP1 on polytene chromosomes are interdependent: both absence and overexpression of HP1 prevent SUUR from chromosomal binding, whereas SUUR overexpression causes redistribution of...]]>
Although the endocannabinoid anandamide is frequently described to act predominantly in the cardiovascular system, the molecular mechanisms of its signaling remained unclear. In human endothelial cells, two receptors for anandamide were found, which were characterized as cannabinoid 1 receptor (CB1R; CNR1) and G-protein-coupled receptor 55 (GPR55). Both receptors trigger distinct signaling pathways. It crucially depends on the activation status of integrins which signaling cascade becomes promoted upon anandamide stimulation. Under conditions of inactive integrins, anandamide initiates CB1R-derived signaling, including Gi-protein-mediated activation of spleen tyrosine kinase (Syk), resulting in NFB translocation. Furthermore, Syk inhibits phosphoinositide 3-kinase (PI3K) that represents a key protein in the transduction of GPR55-originated signaling. However, once integrins are clustered, CB1R splits from integrins and, thus, Syk cannot further inhibit GPR55-triggered signaling resulting in intracellular Ca2+ mobilization from the endoplasmic reticulum (ER) via a PI3K-Bmx-phospholipase C (PLC) pathway and activation of nuclear factor of activated T-cells. Altogether,...]]>
Assembly of the mitotic spindle requires a global change in the activity and constitution of the microtubule-binding-protein array at mitotic onset. An important subset of mitotic microtubule-binding proteins localises to the nucleus in interphase and essentially contributes to spindle formation and function after nuclear envelope breakdown. Here, we used a proteomic approach to selectively identify proteins of this category and revealed 50 poorly characterised human gene products, among them the echinoderm microtubule-associated-protein-like gene product, EML3. Indirect immunofluorescence showed that EML3 colocalises with spindle microtubules throughout all mitotic stages. In interphase, EML3 colocalised with cytoplasmic microtubules and accumulated in interphase nuclei. Using YFP-fusion constructs of EML3, we located a nuclear localisation signal and confirmed the microtubule-binding domain of EML3. Functional analysis of EML3 using time-lapse fluorescence microscopy and detailed end-point analysis of phenotypes after siRNA knockdown demonstrates an important role for EML3 in correct metaphase chromosome alignment. Our proteomic identification...]]>
Several lines of evidence have revealed that ubiquitylation of membrane proteins serves as a signal for endosomal sorting into lysosomes or lytic vacuoles. The hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) interacts with ubiquitylated cargoes through its ubiquitin-interacting-motif domain (UIM domain), and plays an essential early role in endosomal sorting. Here, we show that the C-terminal region of Hrs, which does not contain the UIM domain, can bind to interleukin-2 receptor β (IL-2Rβ). We found a direct interaction between bacterially expressed IL-2Rβ and Hrs in GST pull-down assays, indicating that their binding is independent of ubiquitin. Trafficking and degradation assays revealed that, similarly to wild-type IL-2Rβ, an IL-2Rβ mutant lacking all the cytoplasmic lysine residues is sorted from Hrs-positive early endosomes to LAMP1-positive late endosomes, resulting in degradation of the receptor. By contrast, an IL-2Rβ mutant lacking the Hrs-binding region passes through early endosomes and is mis-sorted to compartments...]]>
Increased expression of BCH-motif-containing molecule at the C-terminal region 1 (BMCC1) correlates with a favourable prognosis in neuroblastoma, but the underlying mechanism remains unknown. We here isolated BNIPXL (BNIP2 Extra Long) as a single contig of the extended, in-vitro-assembled BMCC1. Here, we show that in addition to homophilic interactions, the BNIP2 and Cdc42GAP homology (BCH) domain of BNIPXL interacts with specific conformers of RhoA and also mediates association with the catalytic DH-PH domains of Lbc, a RhoA-specific guanine nucleotide exchange factor (RhoGEF). BNIPXL does not recognize the constitutive active G14V and Q63L mutants of RhoA but targets the fast-cycling F30L and the dominant-negative T19N mutants. A second region at the N-terminus of BNIPXL also targets the proline-rich region of Lbc. Whereas overexpression of BNIPXL reduces active RhoA levels, knockdown of BNIPXL expression has the reverse effect. Consequently, BNIPXL inhibits Lbc-induced oncogenic transformation. Interestingly, BNIPXL can also interact with RhoC,...]]>
The directed cell migration towards a chemotactic source, chemotaxis, involves three complex and interrelated processes: directional sensing, cell polarization and motility. Directional sensing allows migrating eukaryotic cells to chemotax in extremely shallow gradients (<2% across the cell body) of the chemoattractant. Although directional sensing has been observed as spatially restricted responses along the plasma membrane, our understanding of the `compass' of the cell that controls the gradient-induced translocation of proteins during chemotactic movements is still largely lacking. Until now, the dynamical behaviour and mobility of the chemoattractant-receptor molecule has been neglected in models describing the directional sensing mechanisms. Here, we show by single-molecule microscopy an agonist-induced increase in the mobile fraction of cAMP-receptor at the leading edge of chemotacting Dictyostelium discoideum cells. The onset of receptor mobility was correlated to the uncoupling and activation of the G2-protein. A finite-element simulation showed that the increase in mobile fraction of the...]]>
To investigate gene synergism in multistage skin carcinogenesis, the RU486-inducible cre/lox system was employed to ablate Pten function (K14.cre/5Ptenflx) in mouse epidermis expressing activated Fos (HK1.Fos). RU486-treated HK1.Fos/5Ptenflx mice exhibited hyperplasia, hyperkeratosis and tumours that progressed to highly differentiated keratoacanthomas, rather than to carcinomas, owing to re-expression of high p53 and p21WAF levels. Despite elevated MAP kinase activity, cyclin D1 and cyclin E2 overexpression, and increased AKT activity that produced areas of highly proliferative papillomatous keratinocytes, increasing levels of GSK3β inactivation induced a novel p53/p21WAF expression profile, which subsequently halted proliferation and accelerated differentiation to give the hallmark keratosis of keratoacanthomas. A pivotal facet to this GSK3β-triggered mechanism centred on increasing p53 expression in basal layer keratinocytes. This increase in expression reduced activated AKT expression and released inhibition of p21WAF, which accelerated keratinocyte differentiation, as indicated by unique basal layer expression of differentiation-specific keratin K1 alongside premature filaggrin and...]]>
One of the most challenging issues currently facing cell biologists is how signal specificity and compartmentalization is achieved, allowing extracellular stimulation to result in a unique and pre-defined intracellular outcome. For this to occur, intracellular components must be correctly positioned in both space and time. Adaptor molecules, which contain protein-interaction domains, are often involved in the assembly of multimeric complexes that organize intracellular signal-transduction pathways. One such protein is syntenin, a PDZ-domain-containing molecule that has a surprising variety and diversity of interaction partners. Here we assimilate and discuss current data that support a role for syntenin in regulating transmembrane-receptor trafficking, tumour-cell metastasis and neuronal-synapse function.
]]>β-catenin is a crucial mediator of the canonical Wnt-signaling pathway. -catenin is a major β-catenin-binding protein, and overexpressed -catenin can negatively regulate β-catenin activity. Thus, -catenin may be an important modulator of the Wnt pathway. We show here that endogenous -catenin has little impact on the transcriptional activity of β-catenin in developing mammalian organisms. We analyzed β-catenin signaling in mice with conditional deletion of E-catenin (Ctnna1) in the developing central nervous system. This mutation results in brain hyperplasia and we investigated whether activation of β-catenin signaling may be at least partially responsible for this phenotype. To reveal potential quantitative or spatial changes in β-catenin signaling, we used mice carrying a β-catenin-signaling reporter transgene. In addition, we analyzed the expression of known endogenous targets of the β-catenin pathway and the amount and localization of β-catenin in mutant progenitor cells. We found that although loss of E-catenin resulted in disruption of...]]>
Caveolae are membrane domains having caveolin-1 (Cav1) as their main structural component. Here, we determined whether Cav1 affects Ca2+ signaling through the Gq–phospholipase-Cβ (PLCβ) pathway using Fischer rat thyroid cells that lack Cav1 (FRTcav–) and a sister line that forms caveolae-like domains due to stable transfection with Cav1 (FRTcav+). In the resting state, we found that eCFP-Gβ and Gq-eYFP are similarly associated in both cell lines by Forster resonance energy transfer (FRET). Upon stimulation, the amount of FRET between Gq-eYFP and eCFP-Gβ remains high in FRTcav– cells, but decreases almost completely in FRTcav+ cells, suggesting that Cav1 is increasing the separation between Gq-Gβ subunits. In FRTcav– cells overexpressing PLCβ, a rapid recovery of Ca2+ is observed after stimulation. However, FRTcav+ cells show a sustained level of elevated Ca2+. FRET and colocalization show specific interactions between Gq and Cav1 that increase upon stimulation. Fluorescence correlation spectroscopy studies show that the...]]>
Satellite cells are the resident stem cells of adult skeletal muscle. As with all stem cells, how the choice between self-renewal or differentiation is controlled is central to understanding their function. Here, we have explored the role of β-catenin in determining the fate of myogenic satellite cells. Satellite cells express β-catenin, and expression is maintained as they activate and undergo proliferation. Constitutive retroviral-driven expression of wild-type or stabilised β-catenin results in more satellite cells expressing Pax7 without any MyoD – therefore, adopting the self-renewal pathway, with fewer cells undergoing myogenic differentiation. Similarly, preventing the degradation of endogenous β-catenin by inhibiting GSK3β activity also results in more Pax7-positive–MyoD-negative (Pax7+MyoD–) satellite-cell progeny. Consistent with these observations, downregulation of β-catenin using small interfering RNA (siRNA) reduced the proportion of satellite cells that express Pax7 and augmented myogenic differentiation after mitogen withdrawal. Since a dominant-negative version of β-catenin had the same effect as...]]>
Septation and spore formation in fission yeast are compartmentalization processes that occur during the mitotic and meiotic cycles, and that are regulated by the septation initiation network (SIN). In mitosis, activation of Sid2 protein kinase transduces the signal from the spindle pole body (SPB) to the middle of the cell in order to promote the constriction of the actomyosin ring. Concomitant with ring contraction, membrane vesicles are added at the cleavage site to enable the necessary expansion of the cell membrane. In meiosis, the forespore membrane is synthesized from the outer layers of the SPB by vesicle fusion. This membrane grows and eventually engulfs each of the four haploid nuclei. The molecular mechanism that connects the SIN pathway with synthesis of the forespore membrane is poorly understood. Here, we describe a meiosis-specific Sid2-like kinase (Slk1), which is important for the coordination of the growth of the forespore membrane with...]]>
CD44 contributes to inflammation and fibrosis in response to injury. As fibroblast recruitment is critical to wound healing, we compared cytoskeletal architecture and migration of wild-type (CD44WT) and CD44-deficient (CD44KO) fibroblasts. CD44KO fibroblasts exhibited fewer stress fibers and focal adhesion complexes, and their migration was characterized by increased velocity but loss of directionality, compared with CD44WT fibroblasts. Mechanistically, we demonstrate that CD44WT cells generated more active TGFβ than CD44KO cells and that CD44 promotes the activation of TGFβ via an MMP-dependent mechanism. Reconstitution of CD44 expression completely rescued the phenotype of CD44KO cells whereas exposure of CD44KO cells to exogenous active TGFβ rescued the defect in stress fibers and migrational velocity, but was not sufficient to restore directionality of migration. These results resolve the TGFβ-mediated and TGFβ-independent effects of CD44 on fibroblast migration and suggest that CD44 may be critical for the recruitment of fibroblasts to sites of injury...]]>
The adaptor protein Miranda plays a pivotal role in the asymmetric cell division of neuroblasts by asymmetrically segregating key differentiation factors. Miranda localization requires Myosin VI and Myosin II. The apical-then-basal localization pattern of Miranda detected in fixed tissue, and the localization defects in embryos lacking Myosin VI, suggest that Miranda is transported to the basal pole as a Myosin VI cargo. However, the mode and temporal sequence of Miranda localization have not been characterized in live embryos. Furthermore, it is unknown whether Miranda and PON, a second adaptor protein required for asymmetric protein localization, are both regulated by Myosin II. By combining immunofluorescence studies with time-lapse confocal microscopy, we show that Miranda protein forms an apical crescent at interphase, but is ubiquitously localized at prophase in a Myosin-II-dependent manner. FRAP analysis revealed that Miranda protein reaches the basal cortex by passive diffusion throughout the cell, rather than by...]]>
In this study, we report the formation of several cytoplasmic inclusion bodies composed of filamentous actin (F-actin) and generated by experimental treatments using depolymerizing or stabilizing actin toxins in neuronal and non-neuronal mammalian cell lines. The actin-stabilizing toxin jasplakinolide (Jpk) induced, in a microtubule-dependent manner, a single, large F-actin aggregate, which contained β- and -actin, ADF/cofilin, cortactin, and the actin nucleator Arp2/3. This aggregate was tightly associated with the Golgi complex and mitochondria, and was surrounded by vimentin intermediate filaments, microtubules and MAP4. Therefore, the Jpk-induced single, large F-actin aggregate fits the established criteria for being considered an aggresome. Lysosomes and/or autophagic vacuoles, proteasomes and microtubules were found to directly participate in the dissolution of this F-actin aggresome. Finally, the model reported here is simple, highly reproducible and reversible, and it provides an opportunity to test pharmacological agents that interfere with the formation, maintenance and/or disappearance of F-actin-enriched pathological...]]>
Although the contribution of bone marrow-derived cells to regenerating skeletal muscle has been repeatedly documented, there remains considerable debate as to whether this incorporation is exclusively a result of inflammatory cell fusion to regenerating myofibers or whether certain populations of bone marrow-derived cells have the capacity to differentiate into muscle. The present study uses a dual-marker approach in which GFP+ cells were intravenously transplanted into lethally irradiated β-galactosidase+ recipients to allow for simple determination of donor and host contribution to the muscle. FACS analysis of cardiotoxin-damaged muscle revealed that CD45+ bone-marrow side-population (SP) cells, a group enriched in hematopoietic stem cells, can give rise to CD45–/Sca-1+/desmin+ cells capable of myogenic differentiation. Moreover, after immunohistochemical examination of the muscles of both SP- and whole bone marrow-transplanted animals, we noted the presence of myofibers composed only of bone marrow-derived cells. Our findings suggest that a subpopulation of bone marrow SP cells...]]>
In hair follicles, dermal papilla (DP) and dermal sheath (DS) cells exhibit striking levels of plasticity, as each can regenerate both cell types. Here, we show that thrombin induces a phosphoinositide 3-kinase (PI3K)-Akt pathway-dependent acquisition of DS-like properties by DP cells in vitro, involving increased proliferation rate, acquisition of `myofibroblastic' contractile properties and a decreased capacity to sustain growth and survival of keratinocytes. The thrombin inhibitor protease nexin 1 [PN-1, also known as SERPINE2) regulates all those effects in vitro. Accordingly, the PI3K-Akt pathway is constitutively activated and expression of myofibroblastic marker smooth-muscle actin is enhanced in vivo in hair follicle dermal cells from PN-1–/– mice. Furthermore, physiological PN-1 disappearance and upregulation of the thrombin receptor PAR-1 (also known as F2R) during follicular regression in wild-type mice also correlate with such changes in DP cell characteristics. Our results indicate that control of thrombin signaling interferes with hair follicle dermal...]]>
Radial glia play key roles in neuronal migration, axon guidance, and neurogenesis during development of the central nervous system. However, the molecular mechanisms regulating growth and morphology of these extended cells are unknown. We show that ABBA, a novel member of the IRSp53-MIM protein family, is enriched in different types of radial glia. ABBA binds ATP-actin monomers with high affinity and deforms PtdIns(4,5)P2-rich membranes in vitro through its WH2 and IM domains, respectively. In radial-glia-like C6-R cells, ABBA localises to the interface between the actin cytoskeleton and plasma membrane, and its depletion by RNAi led to defects in lamellipodial dynamics and process extension. Together, this study identifies ABBA as a novel regulator of actin and plasma membrane dynamics in radial glial cells, and provides evidence that membrane binding and deformation activity is critical for the cellular functions of IRSp53-MIM-ABBA family proteins.
]]>The Wnt/β-catenin signaling pathway is essential for normal skeletal development because conditional gain or loss of function of β-catenin in cartilage results in embryonic or early postnatal death. To address the role of β-catenin in postnatal skeletal growth and development, Col2a1-ICAT transgenic mice were generated. Mice were viable and had normal size at birth, but became progressively runted. Transgene expression was limited to the chondrocytes in the growth plate and articular cartilages and was associated with decreased β-catenin signaling. Col2a1-ICAT transgenic mice showed reduced chondrocyte proliferation and differentiation, and an increase in chondrocyte apoptosis, leading to decreased widths of the proliferating and hypertrophic zones, delayed formation of the secondary ossification center, and reduced skeletal growth. Isolated primary Col2a1-ICAT transgenic chondrocytes showed reduced expression of chondrocyte genes associated with maturation, and demonstrated that VEGF gene expression requires cooperative interactions between BMP2 and β-catenin signaling. Altogether the findings confirm a crucial...]]>
The Notch signaling pathway appears to perform an important function in inflammation. Here, we present evidence to suggest that lipopolysaccharide (LPS) suppresses Notch signaling via the direct modification of Notch by the nitration of tyrosine residues in macrophages. In the RAW264.7 macrophage cell line and in rat primary alveolar macrophages, LPS was found to inhibit Notch1 intracellular domain (Notch1-IC) transcription activity, which could then be rescued by treatment with N(G)-nitro-l-arginine, a nitric oxide synthase (NOS) inhibitor. Nitric oxide (NO), which was produced in cells that stably express endothelial NOS (eNOS) and brain NOS (bNOS), also induced the inhibition of Notch1 signaling. The NO-induced inhibition of Notch1 signaling remained unchanged after treatment with 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a guanylyl-cyclase inhibitor, and was not found to be mimicked by 8-bromo-cyclic GMP in the primary alveolar macrophages. With regards to the control of Notch signaling, NO appears to have a significant negative influence, via...]]>
Cell-cell junctions are composed of a diverse array of specialized proteins that are necessary for the movement and integrity of epithelia. Scaffolding molecules, such as membrane-associated guanylate kinases (MAGUKs) contain multiple protein-protein interaction domains that integrate these proteins into macromolecular complexes at junctions. We have used structure-function experiments to dissect the role of domains of the Caenorhabditis elegans MAGUK DLG-1, a homolog of Drosophila Discs large and vertebrate SAP97. DLG-1 deletion constructs were analyzed in directed yeast two-hybrid tests as well as in vivo in a dlg-1 null mutant background. Our studies identify novel roles for several key domains. First, the L27 domain of DLG-1 mediates the physical interaction of DLG-1 with its binding partner, AJM-1, as well as DLG-1 multimerization. Second, the PDZ domains of DLG-1 mediate its association with the junction. Third, using dynamic in vivo imaging, we demonstrate that the SH3 domain is required for rapid...]]>
Endocytosis and recycling of membrane proteins are key processes for nutrient uptake, receptor signaling and synaptic transmission. Different steps in these fission and fusion cycles have been proposed to be regulated by physiological changes in plasma membrane (PM) phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] concentration. Here, we use a chemical enzyme-translocation strategy to rapidly reduce PM PtdIns(4,5)P2 levels while monitoring clathrin-mediated endocytosis and recycling. PtdIns(4,5)P2 hydrolysis blocked transferrin receptor endocytosis and led to a marked increase in the concentration of transferrin receptors in the PM, suggesting that endocytosis is more sensitive to changes in PtdIns(4,5)P2 than recycling. Reduction of PM PtdIns(4,5)P2 levels led to a near complete dissociation of Adaptor protein 2 (AP-2) from the PM but had only a small effect on clathrin assembly. This argues that receptor-mediated PtdIns(4,5)P2 reduction preferentially suppresses AP-2-mediated targeting of cargo to endocytic sites rather than the assembly of clathrin coats or recycling of endocytic vesicles.
]]>SNX9, SNX18 and SNX30 constitute a separate subfamily of PX-BAR-containing sorting nexin (SNX) proteins. We show here that most tissues express all three paralogs, and immunoprecipitation and immunofluorescence experiments demonstrated that the SNX9-family proteins act as individual entities in cells. Their SH3 domains displayed a high selectivity for dynamin 2, and the PX-BAR units had the capacity to tubulate membranes when expressed in HeLa cells. As previously described for the PX-BAR domain of SNX9 (SNX9-PX-BAR), purified SNX18-PX-BAR caused liposome tubulation in vitro and had a binding preference for PtdIns(4,5)P2. However, contrary to SNX9, which primarily acts in clathrin-mediated endocytosis at the plasma membrane, endogenous SNX18 localized to AP-1- and PACS1-positive endosomal structures, which were devoid of clathrin and resistant to Brefeldin A. Moreover, a -adaptin recognition motif was defined in a low-complexity region of SNX18, and a complex of endogenous SNX18 and AP-1 could be immunoprecipitated after Brefeldin A...]]>
Bik1p is the budding yeast counterpart of the CLIP-170 family of microtubule plus-end tracking proteins, which are required for dynein localization at plus ends and dynein-dependent spindle positioning. CLIP-170 proteins make up a CAP-Gly microtubule-binding domain, which sustains their microtubule plus-end tracking behaviour. However, in yeast, Bik1p travels towards plus ends as a cargo of the plus-end-directed kinesin Kip2p. Additionally, Kip2p behaves as a plus-end-tracking protein; hence, it has been proposed that Bik1p might track plus ends principally as a cargo of Kip2p. Here, we examined Bik1p localization in yeast strains expressing mutant tubulin lacking the C-terminal amino acid (Glu tubulin; lacking Phe), the interaction of which with Bik1p is severely impaired compared with wild type. In Glu-tubulin strains, despite the presence of robust Kip2p comets at microtubule plus ends, Bik1p failed to track plus ends. Despite Bik1p depletion at plus ends, dynein positioning at the same plus ends...]]>
Similar to most differentiated cells, both neurons and epithelial cells elaborate distinct plasma membrane domains that contain different membrane proteins. We have previously shown that the axonal cell-adhesion molecule L1/NgCAM accumulates on the axonal surface by an indirect transcytotic pathway via somatodendritic endosomes. MDCK epithelial cells similarly traffic NgCAM to the apical surface by transcytosis. In this study, we map the signals in NgCAM required for routing via the multi-step transcytotic pathway. We identify both a previously mapped tyrosine-based signal as a sufficient somatodendritic targeting signal, as well as a novel axonal targeting signal in the cytoplasmic tail of NgCAM. The axonal signal is glycine and serine rich, but only the glycine residues are required for activity. The somatodendritic signal is cis-dominant and needs to be inactivated in order for the axonal signal to be executed. Additionally, we show that the axonal cytoplasmic signal promotes apical targeting in MDCK...]]>
The major-histocompatibility-complex protein UAP56 (BAT1) is a DEAD-box helicase that is deposited on mRNA during splicing. UAP56 is retained on spliced mRNA in an exon junction complex (EJC) or, alternatively, with the TREX complex at the 5' end, where it might facilitate the export of the spliced mRNA to the cytoplasm. Using confocal microscopy, UAP56 was found to be concentrated in RNA-splicing speckled domains of nuclei but was also enriched in adjacent nuclear regions, sites at which most mRNA transcription and splicing occur. At speckled domains, UAP56 was in complexes with the RNA-splicing and -export protein SRm160, and, as measured by FRAP, was in a dynamic binding equilibrium. The application of an in vitro FRAP assay, in which fluorescent nuclear proteins are photobleached in digitonin-extracted cells, revealed that the equilibrium binding of UAP56 in complexes at speckled domains was directly regulated by ATP binding. This was confirmed using a...]]>
Golgins are coiled-coil proteins that have been implicated in the structure and function of the Golgi complex. Here, we identify and characterize a trypanosomal golgin, TbG63, showing that it has a C-terminal membrane anchor and an N-terminus that projects into the cytoplasm. TbG63 in procyclic parasites is localized to the Golgi and interacts with the active, GTP-form of TbRab1A. Overexpression of TbG63 has dramatic effects on Golgi architecture – effects that require the N-terminus – whereas depletion has little, if any, effect on the growth rate. By contrast, in the bloodstream form of the parasite, depletion of TbG63 slows growth, although it has no obvious effect on the transport of a variant surface glycoprotein (VSG) or on Golgi structure. TbG63 might be a useful tool to study the structure and functioning of the Golgi complex.
]]>Several meiosis-specific proteins of Schizosaccharomyces pombe play essential roles in meiotic progression. We report here that a novel meiosis-specific protein kinase, Mug27 (also known as Ppk35), is required for proper spore formation. This kinase is expressed by the mug27+ gene, which is abruptly transcribed after horsetail movement. This transcription is maintained until the second meiotic division. Green fluorescent protein (GFP)-tagged Mug27 appears at the start of prometaphase I, localizes to the spindle pole body (SPB) and then translocates to the forespore membrane (FSM) at late anaphase II. In the mug27 strain, smaller spores are produced compared with those of the mug27+ strain. Moreover, spore viability was reduced by half or more compared with that of the mug27+ strain. The protein-kinase activity of Mug27 appears to be important for its function: the putative kinase-dead Mug27 mutant had similar phenotypes to mug27. Our results here indicate that the Mug27 kinase localizes...]]>
The protozoan phylum Apicomplexa encompasses ~5000 species of obligate intracellular parasites, including those responsible for malaria and toxoplasmosis. Rather than dividing by binary fission, apicomplexans use a remarkable mechanism for replication, assembling daughters de novo within the cytoplasm. Here, we exploit time-lapse microscopy of fluorescent markers targeted to various subcellular structures in Toxoplasma gondii tachyzoites to determine how these unicellular eukaryotes efficiently package a complete set of organelles, maintaining the highly polarized organization necessary for host cell invasion and pathogenesis. Golgi division and elongation of the apicoplast are among the first morphologically observable events, associated with an unusual pattern of centriolar migration. Daughter parasites are assembled on cytoskeletal scaffolding, whose growth proceeds from the apical end, first encapsulating the divided Golgi. Further extension of the cytoskeletal scaffold results in partitioning of the apicoplast, nucleus, endoplasmic reticulum, and finally the mitochondrion, which enters the developing daughters rapidly, but only very...]]>
Correct establishment and maintenance of cell polarity is required for the development and homeostasis of all metazoans. Cell-polarity mechanisms are responsible not only for the diversification of cell shapes but also for regulation of the asymmetric cell divisions of stem cells that are crucial for their correct self-renewal and differentiation. Disruption of cell polarity is a hallmark of cancer. Furthermore, recent evidence indicates that loss of cell polarity is intimately involved in cancer: several crucial cell-polarity proteins are known proto-oncogenes or tumor suppressors, basic mechanisms of cell polarity are often targeted by oncogenic signaling pathways, and deregulation of asymmetric cell divisions of stem or progenitor cells may be responsible for abnormal self-renewal and differentiation of cancer stem cells. Data from in vivo and three-dimensional (3D) cell-culture models demonstrate that tissue organization attenuates the phenotypic outcome of oncogenic signaling. We suggest that polarized 3D tissue organization uses cell-cell and cell-substratum...]]>
Anillin, one of the first factors recruited to the cleavage site during cytokinesis, interacts with actin, myosin II and septins, and is essential for proper organization of the actomyosin contractile ring. We employed affinity-purification methodology coupled with mass spectrometry to identify Anillin-interacting molecules in Drosophila cells. We isolated several actin and myosin proteins, three of the five Drosophila septins and RacGAP50C (Tum), a component of the centralspindlin complex. Using drug and RNA interference (RNAi) treatments we established that F-actin is essential for Anillin cortical localization in prometaphase but not for its accumulation at the cleavage furrow after anaphase onset. Moreover, septins were not recruited to the cleavage site in cells in which Anillin was knocked down by RNAi, but localized to central-spindle microtubules, suggesting that septins travel along microtubules to interact with Anillin at the furrow. Finally, we demonstrate that RacGAP50C is necessary for Anillin accumulation at the furrow...]]>
FERM domain proteins, including talins, ERMs, FAK and certain myosins, regulate connections between the plasma membrane, cytoskeleton and extracellular matrix. Here we show that FrmA, a Dictyostelium discoideum protein containing two talin-like FERM domains, plays a major role in normal cell shape, cell-substrate adhesion and actin cytoskeleton organisation. Using total internal reflection fluorescence (TIRF) microscopy we show that FrmA-null cells are more adherent to substrate than wild-type cells because of an increased number, persistence and mislocalisation of paxillin-rich cell-substrate adhesions, which is associated with decreased motility. We show for the first time that talinA colocalises with paxillin at the distal ends of filopodia to form cell-substrate adhesions and indeed arrives prior to paxillin. After a period of colocalisation, talin leaves the adhesion site followed by paxillin. Whereas talinA-rich spots turnover prior to the arrival of the main body of the cell, paxillin-rich spots turn over as the main body...]]>
The molecular complex containing CARMA proteins, BCL10 and TRAF6 has been identified recently as a key component in the signal transduction pathways that regulate activation of the nuclear factor B (NF-B) transcription factor. Here, we report that the inducible protein A20 negatively regulates these signaling cascades by means of its deubiquitylation activity. We show that A20 perturbs assembly of the complex containing CARMA3, BCL10 and IKK/NEMO, thereby suppressing activation of NF-B. Together, our results further define the molecular mechanisms that control activation of NF-B and reveal a function for A20 in the regulation of CARMA and BCL10 activity in lymphoid and non-lymphoid cells.
]]>The platelet-derived growth factor β receptor (PDGFβR) plays an important role in proliferation and motility of fibroblasts. We have been investigating the effects of sustained PDGFβR activation in mortal human diploid fibroblasts (HDFs), which are typically difficult to transform. We have previously shown that the bovine papillomavirus E5 protein, through its ability to crosslink and constitutively activate the PDGFβR, induces morphological transformation, enhanced growth and loss of contact inhibition (focus formation) in HDFs. Here, we characterized two E5 mutants as being severely defective for focus formation but still competent for enhanced growth, suggesting that proliferation is insufficient for loss of contact inhibition. These E5 mutants were then used in a comparative study to distinguish the PDGFβR signaling intermediates required for the enhanced growth phenotype from those required for focus formation. Our data suggested that a PI 3-kinase (PI3K)-AKT-cyclin D3 pathway, a Grb2-Gab1-SHP2 complex and JNK played a role in...]]>