bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel protein cross-reacting with antibodies against spectrin is localised in the nucleoli of amphibian oocytes
Authors: Carotenuto Rosa , Maturi Giovanna , Infante Vincenzo , Capriglione Teresa , Petrucci Tamara C. , Campanella Chiara
Published: April 2021

Cytoskeletal proteins such as actin and myosin are important constituents of the nucleoplasm. Spectrin is an actin binding protein typically related to plasma membrane; recently, it has been found that it is widespread and forms distinct membrane protein domains in such organelles as the Golgi. In this paper, the large germinal vesicle of amphibian oocytes was chosen as a particularly suitable system to investigate the presence and location of spectrin in the nucleus. We manually isolated the germinal vesicles of both Discoglossus pictus and Xenopus laevis oocytes, and processed them for SDS-PAGE, immunoblotting and immunoprecipitation. By the use of an antibody against the general form of brain β spectrin (βIIΣ1) and of an anti-α brain spectrin (αIIΣ*), a band of 230 kDa was identified as a nuclear spectrin-like molecule. Moreover the 230 kDa protein was extracted from the nuclei by 1 M KCl, similarly to spectrin in other systems. In oocyte sections and nuclear spreads incubated with anti-αIIΣ* and/or anti-βIIΣ1 antibodies, the immunostain was localised in the nucleoplasm and in the outer shell of the round bodies abundantly present in the germinal vesicle. Sections of the same oocytes, stained with a monoclonal antibody against nucleolar fibrillarin and anti-αIIΣ*, showed colocalisation of the two antibodies. It was concluded that, in the germinal vesicle of amphibian oocytes, a spectrin-like molecule is a part of the outer shell of nucleoli. It is hypothesised that spectrin, together with actin, might be instrumental in keeping nucleoli attached to the inner nuclear membrane, as nucleoli migrate during oogenesis to the inner aspect of the nuclear envelope, where they are stably kept until the end of their growth. Furthermore, these results strongly suggest that the 230 kDa band might comprise both an α and a β chain of the same apparent molecular mass, thus constituting a novel form of a spectrin-like molecule.
Cloning of SEC61 homologues from Schizosaccharomyces pombe and Yarrowia lipolytica reveals the extent of functional conservation within this core component of the ER translocation machinery
Authors: Broughton Jon , Swennen Dominique , Wilkinson Barrie M. , Joyet Philippe , Gaillardin Claude , Stirling Colin J.
Published: April 2021

The Sec61 protein is required for protein translocation across the ER membrane in both yeast and mammals and is found in close association with polypeptides during their membrane transit. In Saccharomyces cerevisiae Sec61p is essential for viability and the extent of sequence similarity between the yeast and mammalian proteins (55% sequence identity) suggests that the role of Sec61p in the translocation mechanism is likely to be conserved. In order to further our understanding of the structure and function of Sec61p we have cloned homologues from both Schizosaccharomyces pombe and Yarrowia lipolytica. The S. pombe gene comprises six exons encoding a 479 residue protein which we have immunolocalised to the endoplasmic reticulum. Sequence comparisons reveal that S. pombe Sec61p is 58.6% identical to that of S. cerevisiae. The deduced amino acid sequence of the Y. lipolytica protein shares 68.8% sequence identity with S. cerevisiae Sec61p.

Gene disruption studies have shown that the SEC61 is required for viability in both S. pombe and Y. lipolytica demonstrating that the essential nature of this protein is not unique to S. cerevisiae. Moreover, heterologous complementation studies indicate that the Y. lipolytica SEC61 gene can complement a null mutation in S. cerevisiae. Sequence comparisons between the various eukaryotic Sec61p homologues reveal a number of highly conserved domains, including several transmembrane sequences and the majority of cytosolic loops. These comparisons will provide an important framework for the detailed analysis of interactions between Sec61p and other components of the translocation machinery and between Sec61p and translocating polypeptide chains.
The glucocorticoid receptor and AP-1 are involved in a positive regulation of the muscle regulatory gene myf5 in cultured myoblasts
Authors: Auradé Frédéric , Pfarr Curt M. , Lindon Catherine , Garcia Alphonse , Primig Michael , Montarras Didier , Pinset Christian
Published: April 2021

The muscle regulatory factor, myf5, is involved in the establishment of skeletal muscle precursor cells. Little is known, however, about the control of the expression of the gene encoding this basic helix-loop-helix (bHLH) factor. We have addressed this question in the mouse myogenic cell line, C2, and in a derivative of this cell line where the myf5 gene is the only muscle-specific bHLH factor to be expressed at the myoblast stage. We present evidence that the synthetic glucocorticoid dexamethasone, and the pharmacological agent anisomycin, act synergistically to rapidly upregulate the levels of myf5 transcript and protein. The glucocorticoid antagonist RU 486 abolishes this synergy, demonstrating the involvement of the glucocorticoid receptor. The expression of a dominant negative mutant of c-jun which interferes with the transactivating properties of all AP-1 family members also blocks the induction of myf5 by anisomycin and dexamethasone. An activator of protein kinase C (PKCs), 12-O-tetradecanoyl phorbol 13-acetate (TPA), abolishes the up-regulation of myf5 gene expression by dexamethasone and anisomycin, and its effect is counteracted by an inhibitor of PKCs, GF 109203X. These results point to the possible involvement of PKCs in the negative control of myf5. Evidence that both positive and negative regulation of myf5 transcripts, described here, does not require the fresh synthesis of transcription factors suggests that myf5 may behave like an immediate early gene.
Morphological effects of wortmannin on the endosomal system and GLUT4-containing compartments in rat adipose cells
Authors: Malide Daniela , Cushman Samuel W.
Published: April 2021

Studies using functional and pharmacological approaches have implicated PI 3-kinase as a key intermediate in the glucose transport and GLUT4 translocation responses to insulin. Confocal microscopy was used to investigate the effects of the PI 3-kinase inhibitor wortmannin in isolated rat adipose cells. Independent of insulin, wortmannin induces the appearance of phase-lucent vacuoles containing the endosomal markers TfR, Rab4, M6PR, and cellubrevin. When added before or with insulin, wortmannin blocks insulin-stimulated GLUT4 translocation, but does not influence the basal VAMP2-containing GLUT4 compartment. These results substantiate the concept of a specialized basal GLUT4 compartment mostly distinct from that of the recycling receptors. However, when added after insulin, wortmannin induces a rapid redistribution of GLUT4 from the cell surface into those endosomal-derived vacuoles where the GLUT4 co-localize with TfR, Rab4, cellubrevin, and VAMP2, but not with clathrin, M6PR, Golgi complex markers TGN38-mannosidase II and γ-adaptin, and lysosomal marker lgp-120. Therefore, wortmannin also disrupts insulin-stimulated GLUT4 traffic in the recycling endosomal pathway, at a step distal to the sorting of recycling proteins from late endosomal and TGN markers; wortmannin does not appear to affect internalization from the plasma membrane, and delivery from early to late endosomes or from late endosomes to the TGN. In combination with previous kinetic biochemical studies, these results suggest that: (i) insulin stimulates the exocytosis of GLUT4 through a direct pathway from a specialized basal compartment to the plasma membrane, (ii) during endocytosis in the presence of insulin, GLUT4 is sorted out of the TfR compartment into a separate recycling pathway back to the plasma membrane, and (iii) both of these pathways involve wortmannin sensitive enzymes.
Intracellular Ca2+ signals in Dictyostelium chemotaxis are mediated exclusively by Ca2+ influx
Authors: Nebl T. , Fisher P. R.
Published: April 2021

We measured folate- and cAMP-induced changes in cytoplasmic free calcium concentration ([Ca2+]i) using recombinant aequorin reconstituted in living Dictyostelium cells with coelenterazine-h. The resulting semi-synthetic protein displayed increased sensitivity to Ca2+ allowing accurate measurement of chemoattractant-induced transients at low resting levels. Both folate- and cAMP-induced Ca2+ responses were developmentally regulated, exhibited remarkably similar kinetics and were dependent on the relative rather than the absolute magnitude of increases in attractant concentration. They began after a short delay of 5-10 seconds, leading to a maximum increase in cytosolic calcium concentration after ∼25 seconds and a return to basal level within ∼60 seconds after stimulation. Responses elicited by the two chemoattractants were dose-dependent and saturated between 4 and 20 μM. They depended on the presence of free extracellular calcium ions and were inhibited in a concentration-dependent manner between 10−4 and 10−5 M. In accordance with 45Ca2+-uptake measurements by Milne and Coukell (J. Cell Biol. (1991) 112, 103-110), both responses were also completely inhibited by 15 μM Ruthenium Red, 15 μM carbonylcyanide m-chlorophenylhydrazone (CCCP) and 500 μM gadolinium ions. Under conditions that prohibited influx of Ca2+ from the extracellular medium there were no detectable changes in [Ca2+]i that could be related to a separate release of the ion from intracellular stores. Together, these results show that the Ca2+ signals involved in chemotaxis correlate temporally with actin depolymerization (not polymerization) and are mediated by Ca2+ influx, not IP3-mediated intracellular release.
Altered expression of the α7β1 integrin in human and murine muscular dystrophies
Authors: Hodges B. L. , Hayashi Y. K. , Nonaka I. , Wang W. , Arahata K. , Kaufman S. J.
Published: April 2021

The α7β1 integrin is the primary laminin receptor on skeletal myoblasts and adult myofibers. It has distinct functions during muscle development and contributes to muscle structural integrity. We have studied this integrin in cases where expression of dystrophin or laminin are compromised. Immunofluorescence demonstrates an increase in α7β1 in patients with Duchenne muscular dystrophy and in mdx mice that lack dystrophin. Analysis of RNA from mdx mice and from patients with Duchenne and Becker muscular dystrophies indicates that the increase in the α7β1 integrin is regulated at the level of α7 gene transcription. In contrast, the levels of α7β1 integrin are severely diminished in patients with laminin α2 chain congenital dystrophy muscular dystrophy and in dy/dy mice that also do not make the α2 laminin chain. Analysis of RNA from the hindlimbs of dy/dy mice demonstrated that in the absence of laminin α7 gene transcription is inhibited and limited to specific alternatively spliced isoforms. We suggest that the increased expression of α7β1 integrin in the absence of dystrophin compensates for the reduced dystrophin-mediated linkage of fibers with the basal lamina and modulates the development of pathology associated with these diseases. The decrease in α7β1 integrin and its transcripts in the absence of laminin likely contributes to the severe myopathy that results from laminin α2 chain deficiency and suggests that laminin-2 regulates expression of the α7 integrin gene. The role of the α7β1 integrin in muscle integrity also suggests that compromised expression of this receptor may underlie as yet undefined myopathies.
HSF1 transcription factor concentrates in nuclear foci during heat shock: relationship with transcription sites
Authors: Jolly Caroline , Morimoto Richard I. , Robert-Nicoud Michel , Vourc’h Claire
Published: April 2021

In this paper, we show that upon heat shock, HSF1 concentrates in the nucleus of diploid human fibroblasts in two large foci. The relative distribution of HSF1 nuclear foci and active heat shock protein (hsp) genes was investigated by combining fluorescence in situ hybridization (FISH) for the detection of hsp nuclear transcripts and immunofluorescence for the detection of HSF1. We show that the HSF1 foci are distinct from the sites of hsp70 and hsp90 genes transcription. This is the second report of ploidy-dependent foci of transcription factors that are independent of their specific transcription sites. However, the correlation between the number of HSF1 foci and the ploidy of the cells strongly supports the existence of a specific chromosomal target for HSF1 foci.
A structure/function analysis of Rat7p/Nup159p, an essential nucleoporin of Saccharomyces cerevisiae
Authors: Priore Veronica Del , Heath Catherine V. , Snay Christine A. , MacMillan Amanda , Gorsch Lisa C. , Dagher Suzanne , Cole Charles N.
Published: April 2021

Rat7p/Nup159p is an essential nucleoporin of Saccharomyces cerevisiae originally isolated in a genetic screen designed to identify yeast temperature-sensitive mutants defective in mRNA export. Here we describe a detailed structural-functional analysis of Rat7p/Nup159p. The mutation in the rat7-1 ts allele, isolated in the original genetic screen, was found to be a single base pair change that created a stop codon approximately 100 amino acids upstream of the actual stop codon of this 1,460 amino acid polypeptide, thus eliminating one of the two predicted coiled-coil regions located near the carboxyl terminus of the protein. These coiled-coil regions are essential since an allele lacking both coiled-coil regions was unable to support growth under any conditions. In contrast, no other region of the protein was absolutely required. The SAFG/PSFG repeat region in the central third of the protein was completely dispensable for growth at temperatures between 16°C and 37°C and cells expressing this mutant allele were indistinguishable from wild type. Deletion of the aminoterminal third of the protein, upstream from the repeat region, or the portion between the repeat region and the coiled-coils resulted in temperature-sensitivity, but the two alleles showed distinct phenotypes with respect to the behavior of nuclear pore complexes (NPCs). Taken together, our data suggest that Rat7p/Nup159p is anchored within the NPC through its coiled-coil region and adjacent sequences. In addition, we postulate that the N-terminal third of Rat7p/Nup159p plays an important role in mRNA export.
Post-Golgi biosynthetic trafficking
Authors: Keller Patrick , Simons Kai
Published: April 2021

Eukaryotic cells have developed complex machineries to distribute proteins and lipids from the Golgi complex. Contrary to what has originally been postulated, delivery of proteins to the cell surface is not a simple bulk flow process but involves sorting into distinct pathways from the trans-Golgi network. Here we describe the various routes emerging from the trans-Golgi network in different cell types, and we discuss the mechanisms that mediate sorting into these pathways. While much remains to be learned about these sorting mechanisms, it is apparent that a number of pathways previously believed to be restricted to certain cell types might be used more commonly.
Xenopus Ran-binding protein 1: Molecular interactions and effects on nuclear assembly in Xenopus egg extracts
Authors: Nicolás Francisco J. , Zhang Chuanmao , Hughes Mike , Goldberg Martin W. , Watton Sandra J. , Clarke Paul R.
Published: April 2021

Ran is a nuclear GTPase implicated in nucleocytoplasmic transport, the maintenance of nuclear structure, mRNA processing, and cell cycle regulation. By two-hybrid interaction in yeast, we have identified a Xenopus homologue of Ran-binding protein 1 (RanBP1). Xenopus RanBP1 interacts specifically with the GTP-bound form of Ran and forms complexes in Xenopus egg extracts with Ran, importin-β/karyopherin-β and importin-α/karyopherin-α, but not p10, p120/RanBP7, RanBP2 or other nucleoporins. These complexes may play roles in the recycling of Ran and importins/karyopherins during nucleocytoplasmic transport. Increased concentrations of RanBP1 stabilise an interaction between Ran and RCC1 in egg extracts, inhibiting the exchange activity of RCC1 towards Ran. Under these conditions, the assembly of nuclei from chromatin is dramatically affected: the nuclei do not assemble a lamina and become very small with homogeneously condensed chromatin. They fail to actively import proteins and do not undergo DNA replication. By field emission in-lens scanning electron microscopy, we show that these nuclei have an intact nuclear envelope containing pore complexes, but the envelope is highly convoluted. However, RanBP1 does not directly inhibit nuclear protein import in assembled nuclei. These results suggest that RCC1 and/or Ran have a function early in nuclear assembly that is disrupted by RanBP1.
A nuclear matrix-associated high molecular mass nuclear antigen, HMNA, of chicken and marked decrease of its immunoreactivity during the progression of S phase
Authors: Shimada Kenji , Harata Masahiko , Mizuno Shigeki
Published: April 2021

A hnRNP-free nuclear matrix prepared from chicken MSB-1 cells was used to raise monoclonal antibodies. The monoclonal antibodies 2H3 and 3B7 showed identical non-homogeneous immunofluorescence staining patterns of nuclei in MSB-1 cells and chicken embryonic fibroblasts. In a synchronized culture of MSB-1 cells, the immunore-activity of nuclei with 2H3, but not with 3B7, antibody decreased markedly during the progression of S phase, but returned to the normal level at the next G1 phase. When cells were treated with Triton X-100 prior to fixation with paraformaldehyde or cells were fixed in methanol, nuclei were reactive with 2H3 antibody throughout the S phase. Both 2H3 and 3B7 antibodies recognized a high molecular mass nuclear antigen (HMNA) of approximately 550 kDa, which was associated with the nuclear matrix. HMNA was resistant to extraction with 0.5 M NaCl from the nuclei at the G1/S boundary but became extractable by the end of S phase. A cDNA clone, pBHB36, containing a partial sequence for HMNA was isolated by immunoscreening as a double positive clone with 2H3 and 3B7 antibodies. The deduced 1,150 residue-long sequence of pBHB36 shows no homology with any molecules in the nucleotide and protein sequence databases, and contains different epitope regions for 2H3 and 3B7 antibodies. A possibility of hydrophobic association of HMNA with nuclear protein(s) during the progression of S phase is discussed.
Overexpression of MAP4 inhibits organelle motility and trafficking in vivo
Authors: Chloë Bulinski J. , McGraw Timothy E. , Gruber Dorota , Nguyen H. Lan , Sheetz Michael P.
Published: April 2021

We previously prepared cell lines that inducibly overexpress MAP4, a microtubule (MT)-associated protein widely expressed in non-neuronal cells. Overexpression of either the full-length MAP4 molecule or its MT-binding domain, MTB, stabilized MTs and retarded cell growth, suggesting that overexpressed MAP4 impacts on MT-dependent functions in vivo. To test this hypothesis, we examined MT-based vesicle movements in living cells, using high resolution DIC microscopy. Overexpression of either MAP4 or MTB yielded a dose-dependent reduction in the frequency of MT-dependent organelle movements, relative to control cells. At steady state, both MAP4- and MTB-overexpressing cells showed unusual distributions of transferrin, LDL, dextran, and Golgi elements, as compared to control cells. MAP4 preferentially inhibited receptor-dependent uptake and degradation of LDL, and repositioning of Golgi elements after disruption by the drug, brefeldin A. L-MOCK cells treated with Taxol to stabilize the MTs to an extent equiva-lent to MAP4 overexpression did not show similar inhibition of vesicle motility or organellar trafficking, suggesting that deficits in organelle movements in vivo represent a direct effect of the presence of MAP4 or MTB, rather than an indirect effect of the stabilization of MTs by overexpressed MAP constructs. Our results show that MAP4 has the capacity to affect transport along MTs in vivo; these findings suggest a potential mechanism by which MAP4 could con-tribute to polarization or morphogenesis of cells.
The location of the transport gate in the nuclear pore complex
Authors: Feldherr Carl M. , Akin Debra
Published: April 2021

Signal-mediated nuclear transport is a gated process that occurs through a central transporter element located within the pore complex. The purpose of this investigation was to identify the region of the transporter that functions as the gate; i.e. the region that restricts passive diffusion of macromolecules through the pores. To accomplish this, small gold particles coated with polyethylene glycol (PEG; total particle diameter 40-70 Å) or large PEG-particles (total diameter 110-270 Å) were microinjected into the cytoplasm or nucleoplasm of Xenopus oocytes. Since PEG does not contain either nuclear import or export signals, it is assumed that the particles distribute by simple diffusion. The cells were fixed after 5 or 30 minutes and subsequently examined using TEM. The distribution of the particles located adjacent to and within the pore complexes was then mapped. The results obtained at both 5 and 30 minutes after cytoplasmic injections of small gold were basically the same. The particles readily entered the transporter but, on the average, were approximately 11 times more concentrated in the cytoplasmic half of this structure. The opposite distribution was observed following nuclear injections, i.e. the particles that were located in the transporter were approximately 7 times more numerous in the nuclear half. Our data indicate that there is a single transport gate located in the central domain of the transporter that restricts passive diffusion. The large particles that were injected into the cytoplasm migrated to the surface of the pore complex, but entered the transporter less frequently than small gold. Interestingly, the diffusion of large PEG-particles to the surface of the pores following nuclear injection was greatly restricted; however, this was not the case for similar size particles that were coated with protein containing nuclear export signals (NES). The latter results suggest that the NES is not only required for translocation, but also for migration within the nucleoplasm.
Direct measurement of clathrin-coated vesicle formation using a cell-free assay
Authors: Gilbert Anne , Paccaud Jean-Pierre , Carpentier Jean-Louis
Published: April 2021

Factors controlling the last stages of clathrin-coated vesicle formation were investigated using an assay allowing direct measurement of the detachment of these vesicles from the plasma membrane. Plasma membranes from cultured cells surface-labelled with 125I-α2-macroglobulin (a ligand that preferentially associates with clathrin-coated pits) were isolated by sonication of cells attached to a poly-L-lysine-coated substratum and incubated in the presence of nucleotide(s) ± cytosol. A significant proportion of the membrane-associated radioactivity was released into the incubation medium in sedimentable form (14×106g). The nucleotide and ligand specificities of this process together with the results of a series of biochemical, morphological and gradient analyses, led to the conclusion that measurement of the released sedimentable radioactivity provides a direct estimate of the formation of clathrin-coated vesicles from clathrin-coated pits. A morphological analysis of quick-frozen replicas of these membranes indicated that only the last stages of clathrin-coated vesicle formation were studied in the assay. Taking advantage of this cell-free system, we demonstrate that membrane-associated cytosolic factors and GTP-binding proteins, noteably dynamin, play a crucial role. Moreover, although these events can occur in the absence of ATP and Ca2+, optimal conditions for the formation of clathrin-coated vesicles require the presence of ATP, GTP and cytosol.
An inhibition of gap-junctional communication by cadherins
Authors: Wang Yingjian , Rose Birgit
Published: April 2021

The action of Ca2+-dependent cell-cell adhesion molecules (cadherins) on cell-to-cell channel-mediated intercellular communication was investigated in mouse L and rat Morris hepatoma cells. These cells fail to adhere to one another in aggregation assays and thus seem to lack cell adhesion molecules. Expression of exogenous cadherin induced strong cell-cell adhesion in both cell types, but had opposite effects on communication, causing inhibition in L cells and improvement in hepatoma cells. Both cells express the connexin43 cell-to-cell channel protein. By western blot we found no cadherin-specific changes in connexin43 protein in either cell type, but connexin43 gap junctional plaque staining, i.e. connexin43 localization to cell-cell junctions, was inhibited in L cells and facilitated in hepatoma cells. In addition we found that the inhibitory effect is largely abolished by blockers of glycosylation. Cadherin-cadherin interactions are known to trigger cell type-specific intracellular signal cascades resulting in diverse end effects, and gap junctional communication/plaque formation seems a further example of such cell type-specificity.
Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27
Authors: Guay Johane , Lambert Herman , Gingras-Breton Geneviève , Lavoie Josée N. , Huot Jacques , Landry Jacques
Published: April 2021

We have studied the contribution of the individual kinases of the MAP (mitogen-activated protein) kinase family, including ERK (extracellular-signal regulated kinase), JNK/SAPK (c-JUN NH2-terminal kinase/stress-activated protein kinase) and p38, to activation of the HSP27 (heat shock protein 27) kinase MAPKAP kinase-2/3 and to HSP27 phosphorylation in Chinese hamster CCL39 cells stimulated by either growth factors, cytokines or stressing agents. In vitro assays using fractionated cell extracts or immunoprecipitates indicated that only fractions containing ERK or p38, and not those containing JNK/SAPK, had the capacity to activate MAPKAP kinase-2/3. In vivo, however, it appeared that only p38 is an upstream activator of HSP27 phosphorylation after both stress or growth factor stimulation: expression of an interfering mutant of ras, which blocked the activation of ERK by both types of inducers, had no effect on HSP27 phosphorylation and p38 activation; and the cell-permeant specific inhibitor of p38, SB203580, blocked MAPKAP-kinase2/3 activation and HSP27 phosphorylation. HSP27 has been suggested to have a phosphorylation-activated homeostatic function at the actin cytoskeleton level. This raises the possibility that p38 might be directly involved in mediating actin responses to external stimuli. Accordingly, we observed that a prior activation of p38 increased the stability of the actin microfilaments in cells exposed to cytochalasin D. The effect was dependent on the expression of HSP27 and was totally annihilated by blocking p38 activity with SB203580. The results provide strong support to the idea that activation of p38 during adverse environmental conditions serves a homeostatic function aimed at regulating actin dynamics that would otherwise be destabilized during stress. Its activation during normal agonist stimulation may constitute an additional actin signaling pathway, the importance of which depends on the level of expression of HSP27.
The checkpoint control for anaphase onset does not monitor excess numbers of spindle poles or bipolar spindle symmetry
Authors: Sluder Greenfield , Thompson Elizabeth A. , Miller Frederick J. , Hayes Jeannette , Rieder Conly L.
Published: April 2021

Exit from mitosis in animal cells is substantially delayed when spindle assembly is inhibited, spindle bipolarity is disrupted, or when a monopolar spindle is formed. These observations have led to the proposal that animal cells have a ‘spindle assembly’ checkpoint for the metaphaseanaphase transition that monitors bipolar spindle organization. However, the existence of such a checkpoint is uncertain because perturbations in spindle organization can produce unattached kinetochores, which by themselves are known to delay anaphase onset. In this study we have tested if cells monitor bipolar spindle organization, independent of kinetochore attachment, by analyzing the duration of mitosis in sea urchin zygotes and vertebrate somatic cells containing multipolar spindles in which all kinetochores are attached to spindle poles. We found that sea urchin zygotes containing tripolar or tetrapolar spindles progressed from nuclear envelope breakdown to anaphase onset with normal timing. We also found that the presence of supernumerary, unpaired spindle poles did not greatly prolong mitosis. Observation of untreated PtK1 cells that formed tripolar or tetrapolar spindles revealed that they progressed through mitosis, on average, at the normal rate. More importantly, the interval between the bipolar attachment of the last monooriented chromosome and anaphase onset was normal. Thus, neither of these cell types can detect the presence of gross aberrations in spindle architecture that inevitably lead to aneuploidy. We conclude that animal cells do not have a checkpoint for the metaphase-anaphase transition that monitors defects in spindle architecture independent of the checkpoint that monitors kinetochore attachment to the spindle. For dividing cells in which spindle microtubule assembly is not experimentally compromised, we propose that the completion of kinetochore attachment is the event which limits the time of the metaphase-anaphase transition.
Down-regulation of actin genes precedes microfilament network disruption and actin cleavage during p53-mediated apoptosis
Authors: Guénal Isabelle , Risler Yanick , Mignotte Bernard
Published: April 2021

Inactivation of Simian Virus 40 large T antigen, in cells immortalized with conditional mutants, leads to activation of p53 and apoptosis. We used the mRNA differential display method to identify genes differentially expressed during this process. We found that steady-state levels of mRNA for cytoplasmic actins decreased early during apoptosis. We also showed that, although the steady-state level of the corresponding proteins is not profoundly affected, they are substrates for an interleukin 1-β converting enzyme (ICE)-like protease activated during the process. However, only a very small fraction of actin is proteolysed during the early stages of apoptosis. The microfilament network is affected and non polymerized actin accumulates in apoptotic bodies after the decrease of mRNA levels, but before a significant amount of actin is cleaved. This suggests that down-regulation of actin genes may be involved in microfilament rearrangements during p53-mediated apoptosis.
Role of microtubules and microtubule organizing centers on meiotic chromosome elimination in Sciara ocellaris
Authors: Esteban M.R. , Campos M.C. , Perondini A.L. , Goday C.
Published: April 2021

Spindle formation and chromosome elimination during male meiosis in Sciara ocellaris (Diptera, Sciaridae) has been studied by immunofluorescence techniques. During meiosis I a monopolar spindle is formed from a single polar complex (centrosome-like structure). This single centrosomal structure persists during meiosis II and is responsible for the non-disjunction of the maternal X chromatids. During meiosis I and II non-spindle microtubules are assembled in the cytoplasmic bud regions of the spermatocytes. The chromosomes undergoing elimination during both meiotic divisions are segregated to the bud region where they associate with bundles of microtubules. The presence and distribution of centrosomal antigens in S. ocellaris meiotic spindles and bud regions has been investigated using different antibodies. gamma-Tubulin and centrin are present in the bud as well as in the single polar complex of first meiotic spindle. The results suggest that spermatocyte bud regions contain microtubule-organizing centres (MTOCs) that nucleate cytoplasmic microtubules that are involved in capturing chromosomes in the bud regions. The distribution of actin and myosin in the spermatocytes during meiosis is also reported.
Sorting of actin isoforms in chicken auditory hair cells
Authors: Hofer D. , Ness W. , Drenckhahn D.
Published: April 2021

Most nonmuscle cells of higher vertebrates contain two different actin isoforms, beta- and gamma-cytoplasmic actin. The beta-isoform is with few exceptions the predominant isoform in nonmuscle cells and tissues. Perturbation of the beta:gamma ratio has been shown to affect the organization of bundled actin filaments indicating that the beta- and gamma-genes encode functionally distinct cytoarchitectural information. In the present study we localized by immunostaining beta- and gamma-actin in chicken auditory hair cells. These highly specialized cells serve as model system for studying certain developmental and structural aspects of a complex actin filament system with high architectural precision. We show that gamma-actin is the predominant actin isoform in auditory hair cells with an apparent beta:gamma ratio of approximately 1:2. gamma-Actin is not sorted and occurs in all three actin assemblies of the hair border, i.e. the cores of sensory hairs (stereocilia), the subjacent gel-like actin filament meshwork (cuticular plate) and the zonula adherens ring. In contrast to gamma-actin, the beta-isoform is specifically sorted to the actin filament core bundle of stereocilia that is extensively crosslinked by fimbrin. In view of recent studies showing that L-plastin, the leukocyte homolog of fimbrin, has a higher binding affinity for beta-actin than for gamma-actin, a mechanism is proposed for how hair cells might restrict formation of actin filament bundles to a single cellular site (i.e. the stereocilia). The limited level of expression of beta-actin in hair cells may help to prevent ectopic bundle formation in other cellular compartments.
Human immortalized brown adipocytes express functional β3-adrenoceptor coupled to lipolysis
Authors: Zilberfarb Vladimir , Piétri-Rouxel France , Jockers Ralf , Krief Stéphane , Delouis Claude , Issad Tarik , Strosberg A. Donny
Published: April 2021

Human brown pre-adipocytes were immortalized by microinjection of the genes encoding simian virus 40 T and t antigens under the control of the human vimentin promotor. The transfected pre-adipocytes were cultured for several months with no loss of their morphological characteristics. These cells accumulate lipids and differentiate into adipocytes when treated with insulin, triiodothyronine and dexamethazone. The mRNA of various adipocyte markers was detected by reverse transcriptase-polymerase chain reaction analysis, including hormone-sensitive lipase, lipoprotein lipase, adipsin, glucose transporters 1 and 4, the uncoupling protein (specific of brown adipocytes), and leptin, the product of the ob gene. Pharmacological analyses indicated that the β3-adrenoceptor is the predominant β-adrenoceptor subtype in PAZ6 cells and that this receptor subtype is functionally coupled to adenylate cyclase and lipolysis. The immortalization of human adipocytes will permit pharmacological analysis of the human β3-adrenoceptor function in adipose cells and will allow detailed studies of human adipocyte differentiation.
Cell death of AKR-2B fibroblasts after serum removal: a process between apoptosis and necrosis
Authors: Simm Andreas , Bertsch Gerald , Frank Hermann , Zimmermann Ulrich , Hoppe Jürgen
Published: April 2021

AKR-2B cells disintegrate after serum removal. After a delay of ∼90 minutes, cell death began and reached after six hours a plateau of 40-50% remaining living cells. We used time-lapse video microscopy to monitor dynamic structural changes and to measure the time span of individual cells to die. The first change was the rapid appearance of membrane blebs. Membrane vesicles were rapidly extruded and reintegrated by the cell. This highly dynamic process of an affected cell stopped after 80±20 minutes with its death. Conductivity measurements showed that at that time the membrane was electrically permeable. By using fluorescence double staining with propidium iodide and Hoechst 33258, we show that membrane leakage leading to disintegration is accompanied, and for some cells preceded, by nuclear condensation. The energy state of the intact cells was monitored by measuring the intracellular ATP content which remained high (6 mM) throughout the entire time of investigation. Mitochondrial potential was determined by rhodamine 123 fluorescence in parallel to the measurement of membrane permeability via uptake of propidium iodide and lead to the detection of a cell population that exhibits a high mitochondrial potential and an uptake of propidium iodide indicating a membrane disruption of cells which still have a high energy charge. It is shown by electron microscopy that mitochondria were swollen and damaged in parallel to nuclear condensation. There was no DNA fragmentation as shown by two independent methods. Addition of the ICE-like protease inhibitor tyr-val-ala-aspchloromethylketone immediately after serum starvation lead to an almost complete survival of the cells up to 6 hours. A pronounced protection was still observed after 24 hours, suggesting an involvement of this type of protease in the onset of cell death after serum removal. Apparently, serum withdrawal activates a succession of initial events that are similar to those defined as ‘apoptosis’, i.e. nuclear condensation and membrane blebbing. These steps are, however, accompanied or rapidly followed by cell lysis and disruption of mitochondria, both of which are characteristic of necrosis.
Transfected muscle and non-muscle actins are differentially sorted by cultured smooth muscle and non-muscle cells
Authors: Mounier Nicole , Perriard Jean-Claude , Gabbiani Giulio , Chaponnier Christine
Published: April 2021

We have analyzed by immunolabeling the fate of exogenous epitope-tagged actin isoforms introduced into cultured smooth muscle and non-muscle (i.e. endothelial and epithelial) cells by transfecting the corresponding cDNAs in transient expression assays. Exogenous muscle actins did not produce obvious shape changes in transfected cells. In smooth muscle cells, transfected striated and smooth muscle actins were preferentially recruited into stress fibers. In non-muscle cells, exogenous striated muscle actins were rarely incorporated into stress fibers but remained scattered within the cytoplasm and frequently appeared organized in long crystal-like inclusions. Transfected smooth muscle actins were incorporated into stress fibers of epithelial cells but not of endothelial cells. Exogenous non-muscle actins induced alterations of cell architecture and shape. All cell types transfected by non-muscle actin cDNAs showed an irregular shape and a poorly developed network of stress fibers. β- and γ-cytoplasmic actins transfected into muscle and non-muscle cells were dispersed throughout the cytoplasm, often accumulated at the cell periphery and rarely incorporated into stress fibers. These results show that isoactins are differently sorted: not only muscle and non-muscle actins are differentially distributed within the cell but also, according to the cell type, striated and smooth muscle actins can be discriminated for. Our observations support the assumption of isoactin functional diversity.
Myosin V associates with melanosomes in mouse melanocytes: evidence that myosin V is an organelle motor
Authors: Wu Xufeng , Bowers Blair , Wei Qin , Kocher Bob , Hammer John A.
Published: April 2021

Mice with mutations at the dilute locus exhibit a ‘washed out’ or ‘diluted’ coat color. The pigments that are responsible for the coloration of mammalian hair are produced by melanocytes within a specialized organelle, the melanosome. Each melanocyte is responsible for delivering melanosomes via its extensive dendritic arbor to numerous keratinocytes, which go on to form the pigmented hair shaft. In this study, we show by light immunofluorescence microscopy and immunoelectron microscopy that the myosin V isoform encoded by the dilute locus associates with melanosomes. This association, which was seen in all mouse melanocyte cell lines examined and with two independent myosin V antibodies, was evident not only within completely melanized cells, but also within cells undergoing the process of melanosome biogenesis, where coordinate changes in the distributions of a melanosome marker and myosin V were seen. To determine where myosin V, a known actin-based motor, might play a role in melanosome transport, we also examined the cellular distribution of F-actin. The only region where myosin V and F-actin were both concentrated was in dendrites and dendritic tips, which represent the sole destination for melanosomes and where they accumulate in cultured melanocytes. These results support the idea that myosin V serves as the motor for the outward movement of melanosomes within dendritic extensions, and, together with the available information regarding the phenotype of mutant melanocytes in vitro, argue that coat color dilution is caused by the absense of this myosin V-dependent melanosome transport system.
Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix
Authors: Greiling Doris , Clark Richard A. F.
Published: April 2021

After injury, the wound space is filled with a fibrin/fibronectin clot containing growth factors released by platelets and monocytes. In response to these factors, fibroblasts migrate into the fibrin clot and contribute to the formation of granulation tissue. The functional mechanisms allowing fibroblasts to leave the collagenous matrix of normal connective tissue and invade the provisional matrix of the fibrin clot have not been fully defined. To investigate these mechanisms we established a new in vitro model which simulates specific aspects of early wound healing, that is, the migration of fibroblasts from a threedimensional collagen matrix into a fibrin clot. This transmigration could be induced by physiological concentrations of platelet releasate or platelet-derived growth factor BB (PDGF-BB) in a concentration-dependent manner. At 24 hours irradiated fibroblasts invaded the fibrin gel almost as well as non-irradiated cells, indicating that transmigration was independent of proliferation. Plasminogen and its activators appear to be necessary for invasion of the fibrin clot since protease inhibitors decreased the amount of migration. These serine proteases, however, were not necessary for exit from the collagen gel as fibroblasts migrated out of the collagen gel onto a surface coated with fibrin fibrils even in the presence of inhibitors. Removal of fibronectin (FN) from either the collagen gel or the fibrin gel markedly decreased the number of migrating cells, suggesting that FN provides a conduit for transmigration. Cell movement in the in vitro model was inhibited by RGD peptide, and by monoclonal antibodies against the subunits of the α5β1 and αvβ3 integrin receptor. Thus, the functional requirements for fibroblast transmigration from collagen-rich to fibrin-rich matrices, such as occurs in early wound healing, have been partially defined using an in vitro paradigm of this important biologic process.
Release of ‘caged’ cytosolic Ca2+ Triggers rapid spreading of human neutrophils adherent via integrin engagement
Authors: Pettit Elizabeth J. , Hallett Maurice B.
Published: April 2021

The role of the transient rise in cytosolic free Ca2+ which occurs during neutrophil adhesion and cell spreading is unclear. In order to establish whether such a Ca2+ signal triggers neutrophil shape change, neutrophils co-loaded with fluo3 and Nitr5 (‘caged’ Ca2+) were used with rapidtime confocal laser scanning microscopy. Here we show that the photolytic generation of a Ca2+ rise in neutrophils which were adherent to an integrin-engaging surface, triggered a rapid change in cell morphology, with increases in cell diameter of approximately 175% occurring within 90 seconds of the Ca2+ signal. In non-adhered neutrophils or neutrophils on plain glass, no acceleration of the rate of spreading occurring in response to the release of ‘caged Ca2+’ could be demonstrated. It was concluded that although a rise in cytosolic free Ca2+ was not the sole trigger for neutrophil shape change, with other signals generated by integrin engagement, a rise in cytosolic free Ca2+ accelerated the rate of neutrophil spreading.
Removal and degradation of the free MHC class II p chain in the endoplasmic reticulum requires proteasomes and is accelerated by BFA
Authors: Dusseljee Simone , Wubbolts Richard , Verwoerd Desiree , Tulp Abraham , Janssen Hans , Calafat Jero , Neefjes Jacques
Published: April 2021

We have studied the degradation of the free major histocompatibility complex (MHC) class II β subunit in the ER. Domain swapping experiments demonstrate that both the intraand extracellular domain determine the rate of degradation. Recently, it has been shown that some ER-retained proteins are exported from the ER by the translocon followed by deglycosylation and degradation in the cytosol by proteasomes. Degradation of the β chain follows a different route. The proteasome is involved but inhibition of the proteasome by lactacystin does not result in deglycosylation and export to the cytosol. Instead, the β chain is retained in the ER implying that extraction of the β chain from the ER membrane requires proteasome activity. Surprisingly, brefeldin A accelerates the degradation of the β chain by the proteasome. This suggests that various processes outside the ER are involved in ER-degradation. The ER is the site from where misfolded class II β chains enter a proteasome-dependent degradation pathway.
The path of transcripts from extra-nucleolar synthetic sites to nuclear pores: transcripts in transit are concentrated in discrete structures containing SR proteins
Authors: Iborra F. J. , Jackson D. A. , Cook P. R.
Published: April 2021

The route taken by transcripts from synthetic sites in the nucleus to the cytoplasm has been under scrutiny for years, but details of the pathway remain obscure. A new highresolution method for mapping the pathway is described; HeLa cells are grown in Br-U so that the analogue is incorporated into RNA and exported to the cytoplasm, before Br-RNA is localized by immuno-electron microscopy. After exposure to low concentrations of Br-U for short periods, cells grow normally. Br-RNA is first found in several thousand extra-nucleolar transcription sites or factories (diameter 50–80 nm), before appearing in several hundred new downstream sites (diameter 50–80 nm) each minute; subsequently, progressively more downstream sites become labelled. These sites can be isolated on sucrose gradients as large nuclear ribonucleoprotein particles of ∼200 S. Later, Br-RNA is seen docked ∼200 nm away from ∼20% nuclear pores, before exiting to the cytoplasm. Individual downstream sites are unlikely to contain individual transcripts; rather, results are consistent with groups of transcripts being shipped together from synthetic sites to pores. A subset of SR proteins are excellent markers of this pathway; this subset is concentrated in tens of thousands of sites, which include transcription, downstream and docking sites. Growth in high concentrations of Br-U for long periods is toxic, and Br-RNA accumulates just inside nuclear pores.
Mechanisms of nuclear positioning
Authors: Reinsch Sigrid , Gönczy Pierre
Published: April 2021

The mechanisms underlying two types of microtubuledependent nuclear positioning are discussed. ‘MTOC-dependent nuclear positioning’ occurs when a nucleus is tightly associated with a microtubule organizing center (MTOC). ‘Nuclear tracking along microtubules’ is analogous to the motor-driven motility of other organelles and occurs when the nucleus lacks an associated MTOC. These two basic types of microtubule-dependent nuclear positioning may cooperate in many proliferating ani mal cells to achieve proper nuclear positioning. Microtubule polymerization and dynamics, motor proteins, MAPs and specialized sites such as cortical anchors function to control nuclear movements within cells.