bluuebunny/crossref_metadata_2025 huggingface dataset:
Different 3′ untranslated regions target alternatively processed hu-li tai shao (hts) transcripts to distinct cytoplasmic locations during Drosophila oogenesis
Authors: Whittaker Kellie L. , Ding Dali , Fisher William W. , Lipshitz Howard D.
Published: April 2021

Cytoplasmic mRNA localization is one method by which protein production is restricted to a particular intracellular site. We report here a novel mechanism for localization of transcripts encoding distinct protein isoforms to different destinations. Alternative processing of transcripts produced in the Drosophila ovary by the hu-li tai shao (hts) locus introduces distinct 3′ untranslated regions (3′UTRs) that differentially localize the mRNAs. Three classes of hts mRNA (R2, N32 and N4) are synthesized in the germ line nurse cells and encode proteins with adducin-homologous amino-terminal regions but divergent carboxy-terminal domains. The R2 and N32 classes of mRNA remain in the nurse cells and are not transported into the oocyte. In contrast, the N4 class of transcripts is transported from the nurse cells into the oocyte starting at stage 1, is subsequently localized to the oocyte cortex at stage 8 and then to the anterior pole from stage 9 on. All aspects of N4 transcript transport and localization are directed by the 345-nucleotide(nt)-long 3′ untranslated region (3′UTR). The organization of localization elements in the N4 3′UTR is modular: a 150 nt core is sufficient to direct transport and localization throughout oogenesis. Additional 3′UTR elements function additively together with this core region at later stages of oogenesis to maintain or enhance anterior transcript anchoring. The swallow locus is required to maintain hts transcripts at the anterior pole of the oocyte and functions through the N4 3′UTR. In addition to the three classes of germ line-expressed hts transcripts, a fourth class (R1) is expressed in the somatic follicle cells that surround the germ line cells. This transcript class encodes the Drosophila orthologue of mammalian adducin.
TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells
Authors: Field Helen , Ali Bassam R. S. , Sherwin Trevor , Gull Keith , Croft Simon L. , Field Mark C.
Published: April 2021

The Rab family of small GTPases is a subset of the Ras superfamily. Rabs regulate the flux through individual steps of the intracellular membrane trafficking pathway, such as ER-to-Golgi transport, probably by controlling SNARE complex assembly. In Trypanosoma brucei a number of Rab proteins have been isolated by EST analysis; here we characterise one of these, TbRab2p (originally designated Trab1p), which is a member of the Ypt1p subfamily of Rab proteins. Recombinant TbRab2p is capable of hydrolysing GTP and is post-translationally modified in vitro by addition of a geranylgeranyl prenyl group, properties of an authentic Rab GTPase. Antibodies against recombinant TbRab2p show that in trypanosomes TbRab2p is localised primarily to the endoplasmic reticulum (ER) and colocalises with BiP in wild-type trypanosomes. Over expression of TbRab2p in procyclic form T. brucei results in a cell population having a 40-fold increase in TbRab2p expression. In these cells biosynthesis of procyclin, a secretory pathway glycoprotein, is decreased, accompanied by an increase in general protein biosynthesis, suggesting that excess TbRab2p affects ER function. Heterologous expression of TbRab2p in COS cells resulted in targeting to the pre-Golgi transport intermediate (ERGIC), indicating that the targeting information is conserved between mammals and trypanosomes. Clustal and phylogenetic analyses support assignment of TbRab2p as a Rab2 homologue. In addition, over expression of TbRab2p in trypanosomes results in membrane reorganisation and formation of opaque vesicular structures visible by phase contrast microscopy, consistent with accumulation of ER-derived vesicular structures in cells highly overexpressing TbRab2p. Ultrastructural examination by electron microscopy confirmed the presence of a tubulo-vesicular membrane bound compartment in close proximity to the cis-Golgi, probably equivalent to the ERGIC. TbRab2p is therefore a new ER/ERGIC marker for T. brucei.
Nuclear organisation of NIPP1, a regulatory subunit of protein phosphatase 1 that associates with pre-mRNA splicing factors
Authors: Trinkle-Mulcahy Laura , Ajuh Paul , Prescott Alan , Claverie-Martin Felix , Cohen Stanley , Lamond Angus I. , Cohen Philip
Published: April 2021

Protein phosphatase-1 (PP1) is complexed to many proteins that target it to particular subcellular locations and regulate its activity. Here, we show that ‘nuclear inhibitor of PP1’ (NIPP1), a major nuclear PP1-binding protein, shows a speckled nucleoplasmic distribution where it is colocalised with pre-mRNA splicing factors. One of these factors (Sm) is also shown to be complexed to NIPP1 in nuclear extracts. Immunodepletion of NIPP1 from nuclear extracts, or addition of a ‘dominant negative’ mutant lacking a functional PP1 binding site, greatly reduces pre-mRNA splicing activity in vitro. These findings implicate the NIPP1-PP1 complex in the control of pre-mRNA splicing.
Characterization of a focal adhesion protein, Hic-5, that shares extensive homology with paxillin
Authors: Thomas Sheila M. , Hagel Margit , Turner Christopher E.
Published: April 2021

Paxillin is a focal adhesion scaffolding protein which was originally identified as a substrate of the oncogenic tyrosine kinase, v-src. Paxillin has been proposed to be involved in regulation of focal adhesion dynamics. Two alternatively spliced mouse paxillin cDNAs were cloned and in the process, a paxillin-related protein, Hic-5, was also identified. Cloning and characterization of Hic-5 indicates that this protein shares extensive homology with paxillin. Although Hic-5 was originally characterized as a TGF-β-inducible gene and proposed to be a transcription factor involved in senescence, the studies here demonstrate that Hic-5 is localized to focal adhesion in REF52 cells and can interact with the focal adhesion proteins, Fak, Frnk, and vinculin. In addition, like paxillin, Hic-5 can bind to a negative regulator of Src PTKs, csk but does not bind to the adaptor protein Crk. Like paxillin, localization of this protein to focal adhesions is mediated primarily by the LIM domains; however, sequences outside the LIM domains also play a minor role in focal adhesion targeting. These results suggest that Hic-5 like paxillin could be involved in regulation of focal adhesion dynamics and raise the possibility that Hic-5 and paxillin could have overlapping or opposing functions in the overall regulation of cell growth and differentiation.
Role of collagenase in mediating in vitro alveolar epithelial wound repair
Authors: Planus Emmanuelle , Galiacy Stéphane , Matthay Michael , Laurent Valérie , Gavrilovic Jelena , Murphy Gillian , Clérici Christine , Isabey Daniel , Lafuma Chantal , d’Ortho Marie-Pia
Published: April 2021

Type II pneumocytes are essential for repair of the injured alveolar epithelium. The effect of two MMP collagenases, MMP-1 and MMP-13 on alveolar epithelial repair was studied in vitro. The A549 alveolar epithelial cell line and primary rat alveolar epithelial cell cultures were used. Cell adhesion and cell migration were measured with and without exogenous MMP-1. Wound healing of a cell monolayer of rat alveolar epithelial cell after a mechanical injury was evaluated by time lapse video analysis. Cell adhesion on type I collagen, as well as cytoskeleton stiffness, was decreased in the presence of exogenous collagenases. A similar decrease was observed when cell adhesion was tested on collagen that was first incubated with MMP-1 (versus control on intact collagen). Cell migration on type I collagen was promoted by collagenases.

Wound healing of an alveolar epithelial cell monolayer was enhanced in the presence of exogenous collagenases. Our results suggest that collagenases could modulate the repair process by decreasing cell adhesion and cell stiffness, and by increasing cell migration on type I collagen. Collagen degradation could modify cell adhesion sites and collagen degradation peptides could induce alveolar type II pneumocyte migration. New insights regarding alveolar epithelial cell migration are particularly relevant to investigate early events during alveolar epithelial repair following lung injury.
Induction of antisense Pax-3 expression leads to the rapid morphological differentiation of neuronal cells and an altered response to the mitogenic growth factor bFGF
Authors: Reeves F. C. , Burdge G. C. , Fredericks W. J. , Rauscher F. J. , Lillycrop K. A.
Published: April 2021

Mutations within the Pax-3 gene lead to a range of developmental abnormalities in both humans and mice. In this report, we have investigated the role that Pax-3 plays in neuronal cell development by specifically downregulating Pax-3 expression within a neuronal cell line. This was achieved by stably transfecting the neuronal cell line ND7 with an expression vector in which antisense Pax-3 RNA was produced under the control of the inducible MMTV promoter. In the stable transfectants, we found that the addition of dexamethasone led to the induction of antisense Pax-3 RNA and a rapid downregulation in endogenous Pax-3 protein expression. The decrease in endogenous Pax-3 protein expression corresponded with a dramatic change in the morphology of the cell: the normally rounded ND7 cells exhibited increased cell to substrate adhesion, extended long neurite processes and expressed genes such as snap-25 that are characteristic of a mature neuron. The morphological differentiation induced by a reduction in Pax-3 expression was followed 24-48 hours later by a cessation in cell proliferation. Interestingly the morphological differentiation and cessation in cell proliferation inducted in the cell lines lacking Pax-3 could be reversed by the addition of the mitogenic growth factor EGF but not by bFGF, whose receptor was downregulated in these cells. These results suggest that the expression of Pax-3 is essential to maintain the undifferentiated phenotype of these immature neuronal cells, and in its absence the cells acquire many of the characteristics of a mature neuronal cell. The slow onset of cell cycle arrest in the cells lacking Pax-3 argues against this transcription factor playing a direct role in the regulation of neuronal cell proliferation.
Syndecan-4 and integrins: combinatorial signaling in cell adhesion
Authors: Couchman John R. , Woods Anne
Published: April 2021

It is now becoming clear that additional transmembrane components can modify integrin-mediated adhesion. Syndecan-4 is a transmembrane heparan sulfate proteoglycan whose external glycosaminoglycan chains can bind extracellular matrix ligands and whose core protein cytoplasmic domain can signal during adhesion. Two papers in this issue of JCS demonstrate, through transfection studies, that syndecan-4 plays roles in the formation of focal adhesions and stress fibers. Overexpression of syndecan-4 increases focal adhesion formation, whereas a partially truncated core protein that lacks the binding site for protein kinase Cα and phosphatidylinositol 4,5-bisphosphate acts as a dominant negative inhibitor of focal adhesion formation. Focal adhesion induction does not require interaction between heparan sulfate glycosaminoglycan and ligand but can occur when non-glycanated core protein is overexpressed; this suggests that oligomerization of syndecan-4 plays a major role in signaling from the extracellular matrix in adhesion.
Syndecan-4 core protein is sufficient for the assembly of focal adhesions and actin stress fibers
Authors: Echtermeyer Frank , Baciu Peter C. , Saoncella Stefania , Ge Yimin , Goetinck Paul F.
Published: April 2021

The formation of focal adhesions and actin stress fibers on fibronectin is dependent on signaling through β1 integrins and the heparan sulfate proteoglycan syndecan-4, and we have analyzed the requirement of the glycosaminoglycan chains of syndecan-4 during these events. Chinese hamster ovary cells with mutations in key enzymes of the glycanation process do not synthesize glycosaminoglycan chains and are unable to assemble actin stress fibers and focal contacts when cultured on fibronectin. Transfection of the mutant cells with a cDNA that encodes the core protein of chicken syndecan-4 leads to the production of unglycanated core protein. The overexpression of syndecan-4 core protein in these mutant cells increases cell spreading and is sufficient for these cells to assemble actin stress fibers and focal adhesions similar to wild-type cells seeded on fibronectin and vitronectin matrices. Syndecan-4 core protein colocalizes to focal contacts in mutant cells that have been transfected with the syndecan-4 core protein cDNA. These data indicate an essential role for the core protein of syndecan-4 in the generation of signals leading to actin stress fiber and focal contact assembly.
A dynamic connection between centromeres and ND10 proteins
Authors: Everett Roger D. , Earnshaw William C. , Pluta Ann F. , Sternsdorf Thomas , Ainsztein Alexandra M. , Carmena Mar , Ruchaud Sandrine , Hsu Wei-Li , Orr Anne
Published: April 2021

ND10, otherwise known as nuclear dots, PML nuclear bodies or PODs, are punctate foci in interphase nuclei that contain several cellular proteins. The functions of ND10 have not been well defined, but they are sensitive to external stimuli such as stress and virus infection, and they are disrupted in malignant promyelocytic leukaemia cells. Herpes simplex virus type 1 regulatory protein Vmw110 induces the proteasome-dependent degradation of ND10 component proteins PML and Sp100, particularly the species of these proteins which are covalently conjugated to the ubiquitin-like protein SUMO-1. We have recently reported that Vmw110 also induces the degradation of centromere protein CENP-C with consequent disruption of centromere structure. These observations led us to examine whether there were hitherto undetected connections between ND10 and centromeres. In this paper we report that hDaxx and HP1 (which have been shown to interact with CENP-C and Sp100, respectively) are present in a proportion of both ND10 and interphase centromeres. Furthermore, the proteasome inhibitor MG132 induced an association between centromeres and ND10 proteins PML and Sp100 in a significant number of cells in the G2 phase of the cell cycle. These results imply that there is a dynamic, cell cycle regulated connection between centromeres and ND10 proteins which can be stabilised by inhibition of proteasome-mediated proteolysis.
Distribution of thimet oligopeptidase (E.C. 3.4.24.15) in human and rat testes
Authors: Pineau Charles , McCool Suzan , Glucksman Marc J. , Jégou Bernard , Pierotti Adrian R.
Published: April 2021

Thimet oligopeptidase (TOP:E.C. 3.4.24.15) is a thiol sensitive metalloendopeptidase which is widely distributed and active in most tissues including testis, brain and pituitary. In the median eminence it is postulated to play a role in the degradation of GnRH released from the hypothalamus and thus to modulate LH levels. In the rat and human, the testis is the richest source of TOP activity with levels 3-to 5-fold higher than that of the brain. In order to define the exact localisation of this enzyme within the rat and human testis, the distribution of TOP in the developing and adult gonad was examined in situ and in isolated cells by immunohistochemistry, western blotting and northern blotting analysis.

Ontogeny studies have demonstrated that TOP is detectable by western blotting from 9 days with levels of expression increasing with the age of the animal. Immunolocalisation of the protein in the interstitium was positive from 9 days onwards but was negative within the seminiferous tubules before 35 days of age, whereas TOP mRNA was not detected within the testis until 35 days of age with subsequent stable expression levels up to 90 days. In the adult rat testis, a strong TOP immunoreactivity was observed within seminiferous tubules, in elongating and elongated spermatids and residual bodies. In the interstitial compartment, immunoreactivity was also observed in Leydig cells and throughout the interstitial space. Western blot analyses confirmed the distribution of expression observed using immunochemistry, however Leydig cells display a lower signal than expected from the immunohistochemical data. Northern hybridization showed that the transcript is present in pachytene spermatocytes, early spermatids, and residual bodies, whereas its presence was not observed in Leydig cells probably due to very low levels of expression of the message. Analyses of various human tissue extracts showed that the testis displays the highest levels of TOP mRNA, with immunohistochemical experiments revealing that, as in the rat, the protein is principally expressed in elongated spermatids/residual bodies, and in Leydig cells.

It is concluded that in the human and rat testes, TOP is highly expressed, in particular in post-meiotic germ cells and Leydig cells. The possible involvement of TOP in proteolytic events associated with the process of spermiogenesis and Leydig cell function is currently under investigation.
Studies on the inhibition of endosome fusion by GTPγS-bound ARF
Authors: Jones Arwyn T. , Spiro David J. , Kirchhausen Tomas , Melançon Paul , Wessling-Resnick Marianne
Published: April 2021

Using a cell free assay, we have previously shown that ARF is not required for endosome fusion but that inhibition of fusion by GTPγS is dependent on a cytosolic pool of ARFs. Since ARF is proposed to function in intracellular membrane traffic by promoting vesicle biogenesis, and components of clathrin- and COP-coated vesicles have been localized on endosomal structures, we investigated whether ARF-mediated inhibition of early endosome fusion involves the recruitment or irreversible association of these proteins onto endosomal membranes. We now report that depletion of components of clathrin coated vesicles (clathrin, AP-1 and AP-2) or COPI vesicles (β COP) does not affect the capacity of GTPγS-activated ARF to inhibit endosome fusion. Inhibition of fusion by activated ARF is also independent of endosomal acidification since assays performed in the presence of the vacuolar ATPase inhibitor bafilomycin A1 are equally sensitive to GTPγS-bound ARF. Finally, in contrast to reported effects on lysosomes, we demonstrate that ARF-GTPγS does not induce endosomal lysis. These combined data argue that sequestration of known coat proteins to membranes by activated ARF is not involved in the inhibition of early endosome fusion and that its capacity to inhibit fusion involves other specific interactions with the endosome surface. These results contrast with the mechanistic action of ARF on intra-Golgi transport and nuclear envelope assembly.
Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding
Authors: Schlöndorff Johannes , Blobel Carl P.
Published: April 2021

Metalloprotease-disintegrins (ADAMs) have captured our attention as key players in fertilization and in the processing of the ectodomains of proteins such as tumor necrosis factor α (TNFα), and because of their roles in Notch-mediated signaling, neurogenesis and muscle fusion. ADAMs are integral membrane glycoproteins that contain a disintegrin domain, which is related to snake-venom integrin ligands, and a metalloprotease domain (which can contain or lack a catalytic site). Here, we review and critically discuss current topics in the ADAMs field, including the central role of fertilin in fertilization, the role of the TNFα convertase in protein ectodomain processing, the role of Kuzbanian in Notch signaling, and links between ADAMs and processing of the amyloid-precursor protein.
Substitution of flight muscle-specific actin by human β-cytoplasmic actin in the indirect flight muscle of Drosophila
Authors: Brault Véronique , Reedy Mary C. , Sauder Ursula , Kammerer Richard A. , Aebi Ueli , Schoenenberger Cora-Ann
Published: April 2021

The human β-cytoplasmic actin differs by only 15 amino acids from Act88F actin which is the only actin expressed in the indirect flight muscle (IFM) of Drosophila melanogaster. To test the structural and functional significance of this difference, we ectopically expressed β- cytoplasmic actin in the IFM of Drosophila that lack endogenous Act88F. When expression of the heterologous actin was regulated by ∼1.5 kb of the 5′ promoter region of the Act88F gene, little β-cytoplasmic actin accumulated in the IFM of the flightless transformants. Including Act88F- specific 5′ and 3′ untranslated regions (UTRs) yielded transformants that expressed wild-type amounts of β-cytoplasmic actin. Despite the assembly of β-cytoplasmic actin containing thin filaments to which endogenous myosin crossbridges attached, sarcomere organization was deficient, leaving the transformants flightless. Rather than affecting primarily actin-myosin interactions, our findings suggest that the β-cytoplasmic actin isoform is not competent to interact with other actin-binding proteins in the IFM that are involved in the organization of functional myofibrils.
Trimeric G proteins modulate the dynamic interaction of PKAII with the Golgi complex
Authors: Martín María Esther , Hidalgo Josefina , Vega Francisco M. , Velasco Angel
Published: April 2021

The Golgi complex represents a major subcellular location of protein kinase A (PKA) concentration in mammalian cells where it has been previously shown to be involved in vesicle-mediated protein transport processes. We have studied the factors that influence the interaction of PKA typeII subunits with the Golgi complex. In addition to the cytosol, both the catalytic (Cα) and regulatory (RIIα) subunits of PKAII were detected at both sides of the Golgi stack, particularly in elements of the cis- and trans-Golgi networks. PKAII subunits, in contrast, were practically absent from the middle Golgi cisternae. Cell treatment with either brefeldin A, AlF4- or at low temperature induced PKAII dissociation from the Golgi complex and redistribution to the cytosol. This suggested the existence of a cycle of association/dissociation of PKAII holoenzyme to the Golgi. The interaction of purified RIIα with Golgi membranes was studied in vitro and found not to be affected by brefeldin A while it was sensitive to modulators of heterotrimeric G proteins such as AlF4-, GTPγS, βγ subunits and mastoparan. RIIα binding was stimulated by recombinant, myristoylated Gαi3 subunit and inhibited by cAMP. Pretreatment of Golgi membranes with bacterial toxins known to catalyze ADP-ribosylation of selected Gα subunits also modified RIIα binding. Taken together the data support a regulatory role for Golgi-associated Gα proteins in PKAII recruitment from the cytosol.
Motogenic activity of IGD-containing synthetic peptides
Authors: Schor S. L. , Ellis I. , Banyard J. , Schor A. M.
Published: April 2021

Although the IGD amino acid motif (iso-gly-asp) is a highly conserved feature of the fibronectin type I module, no biological activity has as yet been ascribed to it. We have previously reported that the gelatin-binding domain of fibronectin stimulates the migration of human skin fibroblasts into native, but not denatured, type I collagen substrata. Two IGD-containing type I modules are present within the gelatin-binding domain. The object of this study was to ascertain whether soluble synthetic peptides containing the IGD motif stimulate fibroblast migration. We found that IGD peptides stimulated fibroblast migration in the following order of activity: IGDS (as present in the ninth type I module) > IGDQ (as present in the seventh type I module) > IGD. The scrambled SDGI peptide and the well-characterised RGDS peptide were devoid of motogenic activity. The migratory response of fibroblasts to IGD-containing peptides consisted of two distinct phases: an initial period of peptide-mediated cell activation and a subsequent period of enhanced migration manifest in the absence of further IGD peptide. Cell activation was substratum-independent (occurring equally well on both native and denatured type I collagen substrata), whilst the manifestation of enhanced migration was persistent and substratum-dependent (being evident only by cells adherent to a native collagen substratum). Our data further indicated that cell activation (1) is elicited by a signal transduction cascade occurring within minutes of cell exposure to IGD-containing peptides, (2) is dependent upon integrin αvβ3 functionality, (3) involves the tyrosine phosphorylation of focal adhesion kinase (ppFAK125) and (4) is inhibited by signalling mediated through integrin α5β1. The expression of migration stimulating activity by soluble IGD-containing peptides clearly distinguishes them from their RGD counterparts. This is the first identified biological activity of the highly conserved IGD motif and provides a rational platform for the development of a novel family of therapeutic compounds designed to stimulate cell migration in relevant clinical situations, such as impaired wound healing.
Partial rescue of epithelial phenotype in integrin β4 null mice by a keratin-5 promoter driven human integrin β4 transgene
Authors: Neut Ronald van der , Cachaço Ana Sofia , Thorsteinsdóttir Sólveig , Janssen Hans , Prins Duncan , Bulthuis Jurjen , Valk Martin van der , Calafat Jero , Sonnenberg Arnoud
Published: April 2021

Integrin β4 null mice exhibit extensive epidermal detachment, reminiscent of the human skin blistering disease junctional epidermolysis bullosa associated with pyloric atresia. Hemidesmosomes, the stable adhesion structures of squamous epithelia, are not formed in the absence of α6β4. Null mutant mice die shortly after birth, but apart from their striking epithelial phenotype, no obvious developmental defects have been observed. To elucidate the cause of death in these mice, we generated transgenic mice with a heterologous construct consisting of the squamous epithelial-specific keratin-5 promoter and a human integrin β4 subunit cDNA. The transgene was not expressed in the presence of endogenous β4, probably as a result of competition for a limited pool of α6 subunits. In a β4 null background, however, the transgene was expressed, and its expression pattern followed that of squamous epithelial-specific keratins. These rescued pups appeared healthy and ultrastructural analysis revealed that the interspecies heterodimer α6mouse/β4human was sufficient to trigger the assembly of hemidesmosomes. After a variable period of up to 48 hours after birth these animals began to exhibit haemorrhages at the plantar and palmar areas. We observed the formation of small blisters and found that the transgene was not detectably expressed in this region, which is devoid of hair follicles. The rescued neonates became increasingly cyanotic and died soon after the onset of this phenomenon. We performed a developmental study of the expression of β4 in the complete respiratory tract, but we found no correlation between the spatiotemporal distribution of β4 and the onset of the respiratory insufficiency. It became clear, however, that there was a gradual detachment of squamous epithelia in the oral and nasal cavities which led to obstruction of the respiratory tract, suggesting that in β4 null and rescued mice, neonatal death was a direct consequence of decreased adhesion properties of hairless squamous epithelia, rather than a developmental defect of the lungs.
Timing of calcium and protein synthesis requirements for the first mitotic cell cycle in fertilised Xenopus eggs
Authors: Beckhelling C. , Penny C. , Clyde S. , Ford C.
Published: April 2021

Mitosis is governed by the activity of the M-phase promoting factor (MPF). In some systems, particularly early embryos, transient increases in calcium concentration have been shown to be necessary for mitosis and regulate its timing. By microinjection of the calcium buffer, dibromoBAPTA, into fertilised Xenopus eggs, we have assessed whether calcium events are required to initiate MPF activation and inactivation. Since initial experiments showed that this buffer inhibited protein synthesis, we measured when mitosis and cleavage became independent of translation. We found that, after a period of protein synthesis essential for cleavage, there was a phase during which continued translation affected the timing of cleavage, but was not essential for its occurrence. Measurement of MPF activity in single embryos injected with calcium buffer at different times in the first cell cycle, showed that there were two sensitive periods. The first period of sensitivity blocked MPF activation and coincided with the time at which cleavage became completely independent of protein synthesis. The second sensitive period occurred just before histone kinase activity peaked, and was necessary for kinase inactivation. Preventing inactivation in this way arrested egg extracts in mitosis. These results support the view that transient increases in free calcium concentration contribute to mitotic progression by first triggering MPF activation and subsequently, with elevated MPF activity, inducing its inactivation.
Membrane flow through the Golgi apparatus: specific disassembly of the cis-Golgi network by ATP depletion
Authors: Valle Mercedes del , Robledo Yolanda , Sandoval Ignacio V.
Published: April 2021

Incubation of NRK cells for 30 to 45 minutes with 50 mM 2-deoxy-D-glucose (DOG) in glucose and pyruvate-free medium results in depletion of the cellular ATP pool and in specific disassembly of the cis-Golgi network (CGN), with the stack of Golgi cisternae (SGC) and the trans-Golgi network (TGN) remaining intact and sensitive to BFA. The disassembly of the CGN is mediated by long tubular structures extending outwards from the Golgi complex and involves microtubules. Upon removal of DOG and addition of glucose and pyruvate to the culture medium, the morphology of the CGN is slowly reestablished. Reconstruction of the CGN involves COPI/COPII-positive vesicles that resume the transport of proteins and in particular of CGN membrane proteins out of the ER. Exit of CGN membrane proteins from the ER is insensitive to BFA. In cells pretreated with nocodazole, the CGN membrane proteins are transported to the vicinity of the SGC fragments dispersed throughout the cytoplasm. Ultrastructural studies of cells engaged in the reconstruction of the CGN revealed that the CGN cisterna emerge as tubular structures extending from 0.2-0.3 µm uncoated vesicles prior to their organization on the cis-side of the SGC.
Function of a minus-end-directed kinesin-like motor protein in mammalian cells
Authors: Matuliene Jurgita , Essner Russell , Ryu Jung-Hwa , Hamaguchi Yukihisa , Baas Peter W. , Haraguchi Tokuko , Hiraoka Yasushi , Kuriyama Ryoko
Published: April 2021

CHO2 is a mammalian minus-end-directed kinesin-like motor protein present in interphase centrosomes/nuclei and mitotic spindle fibers/poles. Expression of HA- or GFP-tagged subfragments in transfected CHO cells revealed the presence of the nuclear localization site at the N-terminal tail. This domain becomes associated with spindle fibers during mitosis, indicating that the tail is capable of interaction with microtubules in vivo. While the central stalk diffusely distributes in the entire cytoplasm of cells, the motor domain co-localizes with microtubules throughout the cell cycle, which is eliminated by mutation of the ATP-binding consensus motif from GKT to AAA. Overexpression of the full-length CHO2 causes mitotic arrest and spindle abnormality. The effect of protein expression was first seen around the polar region where microtubule tended to be bundled together. A higher level of protein expression induces more elongated spindles which eventually become disorganized by loosing the structural integrity between microtubule bundles. Live cell observation demonstrated that GFP-labeled microtubule bundles underwent continuous changes in their relative position to one another through repeated attachment and detachment at one end; this results in the formation of irregular number of microtubule focal points in mitotic arrested cells. Thus the primary action of CHO2 appears to cross-link microtubules and move toward the minus-end direction to maintain association of the microtubule end at the pole. In contrast to the full-length of CHO2, overexpression of neither truncated nor mutant polypeptides resulted in significant effects on mitosis and mitotic spindles, suggesting that the function of CHO2 in mammalian cells may be redundant with other motor molecules during cell division.
Epidermal tendon cells require Broad Complex function for correct attachment of the indirect flight muscles in Drosophila melanogaster
Authors: Sandstrom David J. , Restifo Linda L.
Published: April 2021

Drosophila Broad Complex, a primary response gene in the ecdysone cascade, encodes a family of zinc-finger transcription factors essential for metamorphosis. Broad Complex mutations of the rbp complementation group disrupt attachment of the dorsoventral indirect flight muscles during pupal development. We previously demonstrated that isoform BRC-Z1 mediates the muscle attachment function of rbp+ and is expressed in both developing muscle fibers and their epidermal attachment sites. We now report two complementary studies to determine the cellular site and mode of action of rbp+ during maturation of the myotendinous junctions of dorsoventral indirect flight muscles. First, genetic mosaics, produced using the paternal loss method, revealed that the muscle attachment phenotype is determined primarily by the genotype of the dorsal epidermis, with the muscle fiber and the ventral epidermis exerting little or no influence. When the dorsal epidermis was mutant, the vast majority of muscles detached or chose ectopic attachment sites, regardless of the muscle genotype. Conversely, wild-type dorsal epidermis could support attachment of mutant muscles. Second, ultrastructural analysis corroborated and extended these results, revealing defective and delayed differentiation of rbp mutant epidermal tendon cells in the dorsal attachment sites. Tendon cell processes, the stress-bearing links between the epidermis and muscle, were reduced in number and showed delayed appearance of microtubule bundles. In contrast, mutant muscle and ventral epidermis resembled the wild type. In conclusion, BRC-Z1 acts in the dorsal epidermis to ensure differentiation of the myotendinous junction. By analogy with the cell-cell interaction essential for embryonic muscle attachment, we propose that BRC-Z1 regulates one or more components of the epidermal response to a signal from the developing muscle.
Peroxisome degradation in Saccharomyces cerevisiae is dependent on machinery of macroautophagy and the Cvt pathway
Authors: Hutchins Maria U. , Veenhuis Marten , Klionsky Daniel J.
Published: April 2021

Organelle biogenesis and turnover are necessary to maintain biochemical processes that are appropriate to the needs of the eukaryotic cell. Specific degradation of organelles in response to changing environmental cues is one aspect of achieving proper metabolic function. For example, the yeast Saccharomyces cerevisiae adjusts the level of peroxisomes in response to differing nutritional sources. When cells are grown on oleic acid as the sole carbon source, peroxisome biogenesis is induced. Conversely, a subsequent shift to glucose-rich or nitrogen-limiting conditions results in peroxisome degradation. The degradation process, pexophagy, requires the activity of vacuolar hydrolases. In addition, peroxisome degradation is specific. Analyses of cellular marker proteins indicate that peroxisome degradation under these conditions occurs more rapidly and to a greater extent than mitochondrial, Golgi, or cytosolic protein delivery to the vacuole by the non-selective autophagy pathway. To elucidate the molecular mechanism of selective peroxisome degradation, we examined pexophagy in mutants that are defective in autophagy (apg) and the selective targeting of aminopeptidase I to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway. Inhibition of peroxisome degradation in cvt and apg mutants indicates that these pathways overlap and that peroxisomes are delivered to the vacuole by a mechanism that utilizes protein components of the Cvt/autophagy pathways.
Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation
Authors: Plemper Richard K. , Bordallo Javier , Deak Peter M. , Taxis Christof , Hitt Reiner , Wolf Dieter H.
Published: April 2021

The endoplasmic reticulum contains a quality control system that subjects misfolded or unassembled secretory proteins to rapid degradation via the cytosolic ubiquitin proteasome system. This requires retrograde protein transport from the endoplasmic reticulum back to the cytosol. The Sec61 pore, the central component of the protein import channel into the endoplasmic reticulum, was identified as the core subunit of the retro-translocon as well. As import of mutated proteins into the endoplasmic reticulum lumen is successfully terminated, a new targeting mechanism must exist that mediates re-entering of misfolded proteins into the Sec61 pore from the lumenal side de novo. The previously identified proteins Der3p/Hrd1p and, as we show here, Hrd3p of the yeast Saccharomyces cerevisiae, are localised in the endoplasmic reticulum membrane and are essential for the degradation of several substrates of the endoplasmic reticulum degradation machinery. Based on genetic studies we demonstrate that they functionally interact with each other and with Sec61p, probably establishing the central part of the retro-translocon. In the absence of Hrd3p, the otherwise stable protein Der3p/Hrd1p becomes rapidly degraded. This depends on a functional ubiquitin proteasome system and the presence of substrate molecules of the endoplasmic reticulum degradation system. When overexpressed, Der3p/Hrd1p accelerates CPY* degradation in hrd3 cells. Our data suggest a recycling process of Der3p/Hrd1p through Hrd3p. The retro-translocon seems to be build up at least by the Sec61 pore, Der3p/Hrd1p and Hrd3p and mediates both retrograde transport and ubiquitination of substrate molecules.
The heterotrimeric Gi3 protein acts in slow but not in fast exocytosis of rat melanotrophs
Authors: Kreft Marko , Gasman Stéphane , Chasserot-Golaz Sylvette , Kuster Vlasta , Rupnik Marjan , Sikdar Sujit K. , Bader Marie-France , Zorec Robert
Published: April 2021

Besides having a role in signal transduction some trimeric G-proteins may be involved in a late stage of exocytosis. Using immunocytochemistry and confocal microscopy we found that Gi3-protein resides mainly in the plasma membrane, whereas Gi1/2-protein is preferentially associated with secretory granules. To study the function of trimeric Gi3- and Gi1/2-proteins, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. We report here that mastoparan, an activator of trimeric G-proteins, enhances calcium-induced secretory activity in rat melanotrophs. The introduction of synthetic peptides corresponding to the C-terminal domain of the α-subunit of Gi3- and Gi1/2-proteins indicated that Gi3 peptide specifically blocked the mastoparan-stimulated secretory activity, which indicates an involvement of a trimeric Gi3-protein in mastoparan-stimulated secretory activity. Flash photolysis of caged Ca2+-elicited biphasic capacitance increases consisting of a fast and a slower component. Injection of anti-Gi3 antibodies selectively inhibited the slow but not the fast component of secretory activity in rat melanotrophs. We propose that the plasma membrane-bound Gi3-protein may be involved in regulated secretion by specifically controlling the slower kinetic component of exocytosis.
Fission yeast msp1 is a mitochondrial dynamin-related protein
Authors: Pelloquin Laetitia , Belenguer Pascale , Menon Yoann , Gas Nicole , Ducommun Bernard
Published: April 2021

We recently identified Msp1p, a fission yeast Schizosaccharomyces pombe dynamin-related protein, which is essential for the maintenance of mitochondrial DNA. The Msp1p sequence displays typical features of a mitochondrial protein. Here we report in vitro and in vivo data that validate that prediction. We demonstrate that the targeting sequence of Msp1p is processed by recombinant mitochondrial processing peptidase and that Msp1p is imported into S. pombe mitochondria in vitro in the presence of cellular extracts. We show that the first 109 residues of Msp1p encompass a functional peptide signal that is sufficient to direct chimera to mitochondria. Immunofluorescence studies indicate that Msp1p staining colocalises with a mitochondrial marker and electron microscopy shows that the protein is located inside the mitochondria. Mitochondrial enrichment and fractionation further confirm that localisation and show that Msp1p is anchored to the matrix side of the mitochondrial inner membrane. Finally, we report that overexpression of the Msp1 protein results in gross alteration of the mitochondrial structure and function. All together our results suggest that Msp1p is an essential component for mitochondrial maintenance.
Regulation of fibrillin carboxy-terminal furin processing by N-glycosylation, and association of amino- and carboxy-terminal sequences
Authors: Ashworth Jane L. , Kelly Vicky , Rock Matthew J. , Shuttleworth C. Adrian , Kielty Cay M.
Published: April 2021

The molecular mechanisms of fibrillin assembly into microfibrils are poorly understood. In this study, we investigated human fibrillin-1 carboxy-terminal processing and assembly using a recombinant approach. Processing of carboxy-terminal fibrillin-1 was strongly influenced by N-glycosylation at the site immediately downstream of the furin site, and by association with calreticulin. The carboxy terminus of fibrillin-2 underwent less efficient processing than carboxy-terminal fibrillin-1 under identical conditions. Size fractionation of the amino-terminal region of fibrillin-1, and of unprocessed and furin-processed carboxy-terminal region of fibrillin-1, revealed that the amino terminus formed abundant disulphide-bonded aggregates. Some association of unprocessed carboxy-terminal fibrillin-1 was also apparent, but processed carboxy-terminal sequences remained monomeric unless amino-terminal sequences encoded by exons 12-15 were present. These data indicate the presence of fibrillin-1 molecular recognition sequences within the amino terminus and the extreme carboxy-terminal sequence downstream of the furin site, and a specific amino- and carboxy-terminal association which could drive overlapping linear accretion of furin-processed fibrillin molecules in the extracellular space. Differences in processing of the two fibrillin isoforms may reflect differential abilities to assemble in the extracellular space.
FYVE-finger proteins – effectors of an inositol lipid
Authors: Stenmark Harald , Aasland Rein
Published: April 2021

The binding of cytosolic proteins to specific intracellular membranes containing phosphorylated derivatives of phosphatidylinositol (PtdIns) is a common theme in vital cellular processes, such as cytoskeletal function, receptor signalling and membrane trafficking. Recently, several potential effectors of the phosphoinositide 3-kinase product PtdIns 3-phosphate (PtdIns(3)P) have emerged through the observation that a conserved zinc-finger-like domain, the FYVE-finger, binds specifically to this lipid. Here we review current knowledge about the structural basis for the FYVE-PtdIns(3)P interaction, its role in membrane recruitment of proteins and the functions of FYVE-finger proteins in membrane trafficking and other cellular processes.
Relationship between Na+,K+-ATPase and cell attachment
Authors: Contreras R. G. , Shoshani L. , Flores-Maldonado C. , Lázaro A. , Cereijido M.
Published: April 2021

A prolonged ouabain blockade of the Na+,K+-ATPase detaches cells from each other and from the substrate. This suggests the existence of a link between pump (P) and attachment (A). In the present work, we report that MDCK-W cells treated with ouabain increase tyrosine phosphorylation and content of active MAP kinase, redistribute molecules involved in cell attachment (occludin, ZO-1, desmoplakin, cytokeratin, α;-actinin, vinculin and actin), and detach. Genistein and UO126, inhibitors of protein tyrosine kinase and of MAP kinase kinase, respectively, block this detachment. The content of P190Rho-GAP, a GTPase activating protein of the Rho small G-protein subfamily, is increased by ouabain, suggesting that both the Rho/Rac and MAPK pathways are involved.

Another clone of MDCK cells whose Na+,K+-ATPase has a negligible affinity for the drug, show none of the effects described for MDCK-W and remain attached. Ma104 cells, a line that has a high affinity for ouabain and stops pumping, fail to modify phosphorylation, as well as the pattern of distribution of attaching molecules, and remain in the monolayer. Taken together, these results suggest that there is a mechanism (P⟶A) that transduces a blockade of the pump in a detachment of the cell from neighbors and substrate, in which Ma104 cells are faulty.
Occludin proteolysis and increased permeability in endothelial cells through tyrosine phosphatase inhibition
Authors: Wachtel Marco , Frei Karl , Ehler Elisabeth , Fontana Adriano , Winterhalter Kaspar , Gloor Sergio M.
Published: April 2021

Regulation of epithelial and endothelial permeability is essential for proper function of compartmentalized organisms, and tyrosine phosphorylation plays an important role in this process. We analyzed the impact of protein tyrosine phosphatase (PTP) inhibition on the structure of endothelial junctional proteins. In human umbilical vein endothelial cells (HUVECs) the PTP inhibitors phenylarsine oxide (PAO) and pervanadate induced proteolysis of the tight junction protein occludin. Occludin proteolysis was inhibited by the metalloproteinase inhibitor 1,10-phenanthroline (PHEN), but not by inhibitors against other types of proteases. The junctional proteins ZO-1, cadherin and β-catenin were not cleaved. Under conditions of occludin proteolysis, PAO treatment elevated permeability for FITC-dextran. Simultaneous incubation of HUVECs with PAO and PHEN inhibited the rise in permeability by more than 60%. PAO treatment lead to progressive disappearance of occludin from the cell periphery. In contrast, ZO-1, cadherin and β-catenin retained their positions at the sites of intercellular contact. Simultaneous administration of PAO and PHEN greatly prevented the redistribution of occludin. These results demonstrate a selective cleavage of occludin by a metalloproteinase and suggest that this process can contribute to the control of paracellular permeability in endothelial cells.