bluuebunny/crossref_metadata_2025 huggingface dataset:
The synaptic vesicle protein, cysteine-string protein, is associated with the plasma membrane in 3T3-L1 adipocytes and interacts with syntaxin 4
Authors: Chamberlain Luke H. , Graham Margaret E. , Kane Susan , Jackson Joanna L. , Maier Valerie H. , Burgoyne Robert D. , Gould Gwyn W.
Published: April 2021

Adipocytes and muscle cells play a major role in blood glucose homeostasis. This is dependent upon the expression of Glut4, an insulin-responsive facilitative glucose transporter. Glut4 is localised to specialised intracellular vesicles that fuse with the plasma membrane in response to insulin stimulation. The insulin-induced translocation of Glut4 to the cell surface is essential for the maintenance of optimal blood glucose levels, and defects in this system are associated with insulin resistance and type II diabetes. Therefore, a major focus of recent research has been to identify and characterise proteins that regulate Glut4 translocation. Cysteine-string protein (Csp) is a secretory vesicle protein that functions in presynaptic neurotransmission and also in regulated exocytosis from non-neuronal cells. We show that Csp1 is expressed in 3T3-L1 adipocytes and that cellular levels of this protein are increased following cell differentiation. Combined fractionation and immunofluorescence analyses reveal that Csp1 is not a component of intracellular Glut4-storage vesicles (GSVs), but is associated with the adipocyte plasma membrane. This association is stable, and not affected by either insulin stimulation or chemical depalmitoylation of Csp1. We also demonstrate that Csp1 interacts with the t-SNARE syntaxin 4. As syntaxin 4 is an important mediator of insulin-stimulated GSV fusion with the plasma membrane, this suggests that Csp1 may play a regulatory role in this process. Syntaxin 4 interacts specifically with Csp1, but not with Csp2. In contrast, syntaxin 1A binds to both Csp isoforms, and actually exhibits a higher affinity for the Csp2 protein.

The results described raise a number of interesting questions concerning the intracellular targeting of Csp in different cell types, and suggest that the composition and synthesis of GSVs may be different from synaptic and other secretory vesicles. In addition, the interaction of Csp1 with syntaxin 4 suggests that this Csp isoform may play a role in insulin-stimulated fusion of GSVs with the plasma membrane.
Parvin, a 42 kDa focal adhesion protein, related to the α-actinin superfamily
Authors: Olski Thorsten M. , Noegel Angelika A. , Korenbaum Elena
Published: April 2021

We have identified and cloned a novel 42-kDa protein termed α-parvin, which has a single α-actinin-like actin-binding domain. Unlike other members of the α-actinin superfamily, which are large multidomain proteins, α-parvin lacks a rod domain or any other C-terminal structural modules and therefore represents the smallest known protein of the superfamily. We demonstrate that mouse α-parvin is widely expressed as two mRNA species generated by alternative use of two polyadenylation signals. We analyzed the actin-binding properties of mouse α-parvin and determined the Kd with muscle F-actin to be 8.4±2.1 μM. The GFP-tagged α-parvin co-localizes with actin filaments at membrane ruffles, focal contacts and tensin-rich fibers in the central area of fibroblasts. Domain analysis identifies the second calponin homology domain of parvin as a module sufficient for targeting the focal contacts. In man and mouse, a closely related paralogue β-parvin and a more distant relative γ-parvin have also been identified and cloned. The availability of the genomic sequences of different organisms enabled us to recognize closely related parvin-like proteins in flies and worms, but not in yeast and Dictyostelium. Phylogenetic analysis of α-parvin and its para- and orthologues suggests, that the parvins represent a new family of α-actinin-related proteins that mediate cell-matrix adhesion.
Use of abnormal preprophase bands to decipher division plane determination
Authors: Granger Cheryl L. , Cyr Richard J.
Published: April 2021

Many premitotic plant cells possess a cortical preprophase band of microtubules and actin filaments that encircles the nucleus. In vacuolated cells, the preprophase band is visibly connected to the nucleus by a cytoplasmic raft of actin filaments and microtubules termed the phragmosome. Typically, the location of the preprophase band and phragmosome corresponds to, and thus is thought to influence, the location of the cell division plane. To better understand the function of the preprophase band and phragmosome in orienting division, we used a green fluorescent protein-based microtubule reporter protein to observe mitosis in living tobacco bright yellow 2 cells possessing unusual preprophase bands. Observations of mitosis in these unusual cells support the involvement of the preprophase band/phragmosome in properly positioning the preprophase nucleus, influencing spindle orientation such that the cytokinetic phragmoplast initially grows in an appropriate direction, and delineating a region in the cell cortex that attracts microtubules and directs later stages of phragmoplast growth. Thus, the preprophase band/phragmosome appears to perform several interrelated functions to orient the division plane. However, functional information associated with the preprophase band is not always used or needed and there appears to be an age or distance-dependent character to the information. Cells treated with the anti-actin drug, latrunculin B, are still able to position the preprophase nucleus suggesting that microtubules may play a dominant role in premitotic positioning. Furthermore, in treated cells, spindle location and phragmoplast insertion are frequently abnormal suggesting that actin plays a significant role in nuclear anchoring and phragmoplast guidance. Thus, the microtubule and actin components of the preprophase band/phragmosome execute complementary activities to ensure proper orientation of the division plane.
Microtubules in the fungal pathogen Ustilago maydis are highly dynamic and determine cell polarity
Authors: Steinberg Gero , Wedlich-Söldner Roland , Brill Marianne , Schulz Irene
Published: April 2021

Many fungal pathogens undergo a yeast-hyphal transition during their pathogenic development that requires rearrangement of the cytoskeleton, followed by directed membrane traffic towards the growth region. The role of microtubules and their dynamic behavior during this process is not well understood. Here we set out to elucidate the organization, cellular role and in vivo dynamics of microtubules in the dimorphic phytopathogen Ustilago maydis. Hyphae and unbudded yeast-like cells of U. maydis contain bundles of spindle pole body-independent microtubules. At the onset of bud formation two spherical tubulin structures focus microtubules towards the growth region, suggesting that they support polar growth in G2, while spindle pole body-nucleated astral microtubules participate in nuclear migration in M and early G1. Conditional mutants of an essential α-tubulin gene from U. maydis, tub1, confirmed a role for interphase microtubules in determination of cell polarity and growth. Observation of GFP-Tub1 fusion protein revealed that spindle pole body-independent and astral microtubules are dynamic, with elongation and shrinkage rates comparable to those found in vertebrate systems. In addition, very fast depolymerization was measured within microtubule bundles. Unexpectedly, interphase microtubules underwent bending and rapid translocations within the cell, suggesting that unknown motor activities participate in microtubule organization in U. maydis.

Movies available on-line: http://www.biologists.com/JCS/movies/jcs1792.html
Molecular architecture of the amplified nucleoli of Xenopus oocytes
Authors: Mais Christine , Scheer Ulrich
Published: April 2021

An understanding of the functional organization of nucleoli, the sites of ribosome biosynthesis, is limited by the present uncertainty about the topological arrangement of the transcribing rRNA genes. Since studies with ‘standard’ nucleoli from somatic cells produced conflicting results, we have examined the amplified nucleoli of Xenopus oocytes. These nucleoli are unique in that they contain high copy numbers of rRNA genes, are not attached to chromosomes, lack non-ribosomal DNA and can be examined in light microscopic spread preparations of nuclear contents. By immunostaining and confocal microscopy we show that in growing stage IV oocytes the sites of rDNA are surrounded by the dense fibrillar component. The rDNA is actively transcribed as revealed by BrUTP injection into oocytes and localization of components of the nucleolar transcription machinery (RNA polymerase I and the transcription factor UBF). At the ultrastructural level, the rDNA sites correlate with the fibrillar centers of amplified nucleoli fixed in situ. The results provide clear evidence that the transcriptionally active rRNA genes are confined to the fibrillar centers of the oocyte nucleoli and open the possibility to analyze the protein composition of almost native, transcriptionally highly active nucleolar chromatin by immunofluorescence microscopy.
The(f)art of criticism
Authors: Marshall Wallace
Published: April 2021

An occasional column, in which Caveman and other troglodytes involved in cell science emerge to share their views on various aspects of life-science research. Messages for Caveman and other contributors can be left at caveman@biologists.com.

Any correspondence may be published in forthcoming issues.
Diversity in phagocytic signalling
Authors: Greenberg Steven
Published: April 2021

Phagocytosis is a phylogenetically ancient process triggered by particulate ligands, such as bacteria, fungi and environmental particles. The spectrum of phagocytically competent cell types and receptors is diverse, but many of the basic processes of cytoskeletal rearrangement and membrane trafficking that accompany phagocytosis are conserved. Insights from studies of lower eukaryotes, such as Dictyostelium, Caenorhabditis elegans and Drosophila have contributed greatly to our understanding of the phagocytic pathway. Here, I focus on only a few examples of phagocytosis and bacterial invasion in mammalian cells, in which specific receptors have been identified and signal transduction pathways have been mapped. Owing to space constraints, the roles of lipid microdomains in phagocytosis and clearance of apoptotic bodies are not covered. The examples provided below emphasize the ability of the host cell to utilize different surface receptors to engage the phagocytic machinery.
Phosphoinositides in membrane traffic at the synapse
Authors: Cremona Ottavio , Camilli Pietro De
Published: April 2021

Inositol phospholipids represent a minor fraction of membrane phospholipids; yet they play important regulatory functions in signaling pathways and membrane traffic. The phosphorylated inositol ring can act either as a precursor for soluble intracellular messengers or as a binding site for cytosolic or membrane proteins. Hence, phosphorylation-dephosphorylation of phosphoinositides represents a mechanism for regulation of recruitment to the membrane of coat proteins, cytoskeletal scaffolds or signaling complexes and for the regulation of membrane proteins. Recent work suggests that phosphoinositide metabolism has an important role in membrane traffic at the synapse. PtdIns(4,5)P2 generation is implicated in the secretion of at least a subset of neurotransmitters. Furthermore, PtdIns(4,5)P2 plays a role in the nucleation of clathrin coats and of an actin-based cytoskeletal scaffold at endocytic zones of synapses, and PtdIns(4,5)P2 dephosphorylation accompanies the release of newly formed vesicles from these interactions. Thus, the reversible phosphorylation of inositol phospholipids may be one of the mechanisms governing the timing and vectorial progression of synaptic vesicle membranes during their exocytic-endocytic cycle.
Cdk5 mediates changes in morphology and promotes apoptosis of astrocytoma cells in response to heat shock
Authors: Gao Chun , Negash Sewite , Wang Hwai-Shi , Ledee Dolena , Guo Hongtao , Russell Paul , Zelenka Peggy
Published: April 2021

The cyclin-dependent kinase member, Cdk5, is expressed in a variety of cell types, but neuron-specific expression of its activator, p35, is thought to limit its activity to neurons. Here we demonstrate that both Cdk5 and p35 are expressed in the human astrocytoma cell line, U373. Cdk5 and p35 are present in the detergent-insoluble cytoskeletal fraction of this cell line and Cdk5 localizes to filopodia and vinculin-rich regions of cell-matrix contact in lamellopodia. When exposed to a 46°C heat shock, U373 cells change shape, lose cell-matrix contacts and show increased levels of apoptosis. To test whether Cdk5 activation might play a role in these events, U373 cells were stably transfected with histidine-tagged or green fluorescent protein-tagged constructs of Cdk5 or a dominant negative mutation, Cdk5T33. Under normal growth conditions, growth characteristics of the stably transfected lines were indistinguishable from untransfected U373 cells and Cdk5 localization was not changed. However, when subjected to heat shock, cells stably transfected with Cdk5-T33 remained flattened, showed little loss of cell-matrix adhesion, and exhibited significantly lower levels of apoptosis. In contrast, cells that overexpressed wild-type Cdk5 showed morphological changes similar to those seen in untransfected U373 cells in response to heat shock and had significantly higher levels of apoptosis. Heat-shocked cells showed changes in p35 mobility and stability of the Cdk5/p35 complex consistent with endogenous Cdk5 activity. Together these findings suggest that endogenous Cdk5 activity may play a key role in regulating morphology, attachment, and apoptosis in U373 cells, and raise the possibility that Cdk5 may be a general regulator of cytoskeletal organization and cell adhesion in both neuronal and non-neuronal cells.
Rap1 is involved in cell stretching modulation of p38 but not ERK or JNK MAP kinase
Authors: Sawada Yasuhiro , Nakamura Kozo , Doi Koichi , Takeda Kohsuke , Tobiume Kei , Saitoh Masao , Morita Keiichi , Komuro Issei , Vos Kurt De , Sheetz Michael , Ichijo Hidenori
Published: April 2021

Mechanical force or mechanical stress modulates intracellular signal pathways, including the mitogen-activated protein kinase (MAP kinase) cascades. In our system, cell stretching activated and cell contraction inactivated all three MAP kinase pathways (MKK1/2-extracellular signal-regulated kinase (ERK), MKK4 (SEK1)-cJun N-terminal kinase (JNK) and MKK3/6-p38 pathways). However, little is known about the molecular mechanisms that link the mechanical force to the MAP kinase cascades. To test whether Ras and Rap1 are possible components in the stretch-activated MAP kinase pathways, we examined if Ras and Rap1 were activated by cell stretching and if inhibition of their activity decreased the stretch-enhanced MAP kinase activity. Rap1 was activated by cell stretching and inactivated by cell contraction, whereas Ras was inactivated by cell stretching and activated by cell contraction. Rap1GapII and SPA-1, downregulators of Rap1 activity, decreased the stretch-enhanced p38 activity, whereas a dominant-negative mutant of Ras (RasN17) did not inhibit the stretch-initiated activation of MAP kinases. Furthermore, overexpression of Rap1 enhanced p38 activity but not ERK or JNK activity. These results indicate that Rap1 is involved in transducing the stretch-initiated signal to the MKK3/6-p38 pathway, but not to the MEK1/2-ERK or the MKK4 (SEK1)/MKK7-JNK pathway. Thus, Rap1 plays a unique role in force-initiated signal transduction.
VEGF transiently disrupts gap junctional communication in endothelial cells
Authors: Suarez S. , Ballmer-Hofer K.
Published: April 2021

Vascular endothelial growth factor, VEGF, stimulates angiogenesis by directly acting on endothelial cells. The effects of VEGF are mediated by two tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR) that are highly related to receptors of the platelet derived growth factor (PDGF) receptor family. We are interested in early signalling events downstream from VEGF receptors that affect blood vessel homeostasis.

Endothelial cells form multiple types of cell-cell junctions that are required for cellular organization into complex networks. These junctions also regulate communication among adjacent cells. Stimulation by various growth factors such as epidermal growth factor (EGF) or PDGF has been shown to disrupt cell-cell junctions, consequently affecting cell-to-cell communication. We investigated gap junctional communication (GJC) by monitoring the transfer of a low molecular mass fluorescent tracer molecule between adjacent cells using immunofluorescence microscopy. VEGF maximally blocked GJC 15 minutes after growth factor administration. The cells resumed communication via gap junctions within 1-2 hours after treatment. This early effect of VEGF on communication correlated with changes in the phosphorylation state of one of the proteins involved in gap junction formation, connexin 43 (Cx43). The signalling mechanisms involved in this phenomenon depend on activation of VEGFR-2, impinge on a tyrosine kinase of the Src family and activate the Erk family of MAP kinases. The function of VEGF-mediated disruption of GJC might be to restrict an increase in endothelium permeability to the environment affected by local injury to blood vessels.
Oligomerisation of G-protein-coupled receptors
Authors: Milligan Graeme
Published: April 2021

A range of approaches have recently provided evidence that G-protein-coupled receptors can exist as oligomeric complexes. Both homo-oligomers, comprising multiple copies of the same gene product, and hetero-oligomers containing more than one receptor have been detected. In several, but not all, examples, the extent of oligomerisation is regulated by the presence of agonist ligands, and emerging evidence indicates that receptor hetero-oligomers can display distinct pharmacological characteristics. A chaperonin-like role for receptor oligomerisation in effective delivery of newly synthesised receptors to the cell surface is a developing concept, and recent studies have employed a series of energy-transfer techniques to explore the presence and regulation of receptor oligomerisation in living cells. However, the majority of studies have relied largely on co-immunoprecipitation techniques, and there is still little direct information on the fraction of receptors existing as oligomers in intact cells.
Recruitment of β-catenin to cadherin-mediated intercellular adhesions is involved in myogenic induction
Authors: Goichberg Polina , Shtutman Michael , Ben-Ze’ev Avri , Geiger Benjamin
Published: April 2021

Cadherin-mediated cell adhesion is involved in muscle differentiation from early stages of myogenic induction to late stages of myoblast interaction and fusion. β-Catenin is a major constituent of cadherin-based adherens junctions and also serves as a signal transduction molecule that regulates gene expression during development. In this study, we explored the involvement of β-catenin in myogenic differentiation. We show here that shortly after a switch from growth to differentiation medium, β-catenin translocates to cell-cell junctions and its levels increase. We further show that elevation of β-catenin levels, induced either by inhibition of its breakdown, using LiCl, or by its overexpression, suppresses the formation of adherens junctions, resulting in a sharp decline in myogenin expression and an arrest of myogenic progression. Recruitment of β-catenin to adherens junctions after transfection with N-cadherin restores myogenin expression in the transfected cells. These results suggest that increased cadherin-mediated adhesion and translocation of β-catenin to adherens junctions are involved in activating the early steps of myogenic differentiation.
The GTPase Rac1 selectively regulates Salmonella invasion at the apical plasma membrane of polarized epithelial cells
Authors: Criss Alison K. , Ahlgren Dawn M. , Jou Tzuu-Shuh , McCormick Beth A. , Casanova James E.
Published: April 2021

The bacterial pathogen Salmonella typhimurium colonizes its animal hosts by inducing its internalization into intestinal epithelial cells. This process requires reorganization of the actin cytoskeleton of the apical plasma membrane into elaborate membrane ruffles that engulf the bacteria. Members of the Rho family of small GTPases are critical regulators of actin structure, and in nonpolarized cells, the GTPase Cdc42 has been shown to modulate Salmonella entry. Because the actin architecture of epithelial cells is organized differently from that of nonpolarized cells, we examined the role of two Rho family GTPases, Cdc42 and Rac1, in invasion of polarized monolayers of MDCK cells by S. typhimurium. Surprisingly, we found that endogenous Rac1, but not Cdc42, was activated during bacterial entry at the apical pole, and that this activation required the bacterial effector protein SopE. Furthermore, expression of dominant inhibitory Rac1 but not Cdc42 significantly inhibited apical internalization of Salmonella, indicating that Rac1 activation is integral to the bacterial entry process. In contrast, during basolateral internalization, both Cdc42 and Rac1 were activated; however, neither GTPase was required for entry. These findings, which differ significantly from previous observations in nonpolarized cells, indicate that the host cell signaling pathways activated by bacterial pathogens may vary with cell type, and in epithelial tissues may further differ between plasma membrane domains.
Analysis of R-Ras signalling pathways
Authors: Self Annette J. , Caron Emmanuelle , Paterson Hugh F. , Hall Alan
Published: April 2021

R-Ras has a high degree of sequence homology to Ras and to other members of the Ras subfamily including Rap, TC21 and M-Ras. Activated versions of Ras and TC21 are highly transforming in a variety of cell lines and mutated forms of both proteins have been found in human tumours. R-Ras interacts with many of the same proteins as Ras and TC21, including c-Raf1, and can induce transformed foci, although this activity is weak compared to Ras and appears to be cell-type specific. Here, we have investigated R-Ras signalling pathways in a variety of cell types. We find that microinjection of activated R-Ras into quiescent fibroblasts stimulates cell cycle progression through G1 phase and subsequent DNA synthesis. However, unlike Ras, R-Ras does not activate the ERK MAP kinase pathway nor does it activate the JNK or p38/Mpk2 MAP kinase pathways. Microinjection of R-Ras into PC12 cells does not induce terminal differentiation, but instead causes extensive cell spreading, consistent with R-Ras having a role in integrin activation. Finally, in a macrophage cell line, R-Ras activates the αMβ2 integrin via the small GTPase Rap1, leading to phagocytosis of opsonized red blood cells, whereas Ras does not. These results indicate that R-Ras has an important role in the regulation of cell growth and adhesion, but that this is mediated through downstream signals distinct from those used by Ras.
The Tem1 small GTPase controls actomyosin and septin dynamics during cytokinesis
Authors: Lippincott John , Shannon Katie B. , Shou Wenying , Deshaies Raymond J. , Li Rong
Published: April 2021

Cytokinesis in budding yeast involves an actomyosin-based ring which assembles in a multistepped fashion during the cell cycle and constricts during cytokinesis. In this report, we have investigated the structural and regulatory events that occur at the onset of cytokinesis. The septins, which form an hour-glass like structure during early stages of the cell cycle, undergo dynamic rearrangements prior to cell division: the hourglass structure splits into two separate rings. The contractile ring, localized between the septin double rings, immediately undergoes contraction. Septin ring splitting is independent of actomyosin ring contraction as it still occurs in mutants where contraction fails. We hypothesize that septin ring splitting may remove a structural barrier for actomyosin ring to contract. Because the Tem1 small GTPase (Tem1p) is required for the completion of mitosis, we investigated its role in regulating septin and actomyosin ring dynamics in the background of the net1-1 mutation, which bypasses the anaphase cell cycle arrest in Tem1-deficient cells. We show that Tem1p plays a specific role in cytokinesis in addition to its function in cell cycle progression. Tem1p is not required for the assembly of the actomyosin ring but controls actomyosin and septin dynamics during cytokinesis.
Cell-specific targeting of caveolin-1 to caveolae, secretory vesicles, cytoplasm or mitochondria
Authors: Li Wei-Ping , Liu Pingsheng , Pilcher Brian K. , Anderson Richard G. W.
Published: April 2021

In commonly used tissue culture cells, caveolin-1 is embedded in caveolae membranes. It appears to reach this location after being cotranslationally inserted into ER membranes, processed in the Golgi and shipped to the cell surface. We now report that caveolae are not the preferred location for caveolin-1 in all cell types. Skeletal muscle cells and keratinocytes target caveolin-1 to the cytosol while in exocrine and endocrine cells it accumulates in the secretory pathway. We also found that airway epithelial cells accumulate caveolin-1 in modified mitochondria. The cytosolic and the secreted forms appear to be incorporated into a soluble, lipid complex. We conclude that caveolin-1 can be targeted to a variety of intracellular destinations, which suggests a novel mechanism for the intracellular traffic of this protein.
Modulation of intracellular transport of acidic fibroblast growth factor by mutations in the cytoplasmic receptor domain
Authors: Citores Lucía , Khnykin Denis , Sørensen Vigdis , Wesche Jørgen , Klingenberg Olav , Więdłocha Antoni , Olsnes Sjur
Published: April 2021

Endocytic uptake and intracellular transport of acidic fibroblast growth factor (aFGF) was studied in cells transfected with FGF receptor 4 with mutations in the cytoplasmic part. Endocytic uptake in HeLa cells was reduced but not abolished when the tyrosine kinase of the receptor was inactivated by mutations or deletions. The tyrosine kinase-dependent endocytosis of aFGF was prevented by the expression of a dominant negative dynamin mutant that blocks endocytosis from coated pits and caveolae. However, more than half of the total endocytic uptake of aFGF was not affected under these conditions, indicating an endocytic uptake mechanism not involving coated pits or caveolae. Mutation or deletion of a putative caveolin-binding sequence did not prevent the localization of part of the receptors to a low density, caveolin-containing subcellular fraction. Whereas wild-type receptor transfers the growth factor from early endosomes to the recycling compartment, kinase negative, full length receptors were inefficient in this respect and the growth factor instead accumulated in lysosomes. By contrast, when most of the intracellular part of the receptor, including the kinase domain, was removed, aFGF was transported to the recycling compartment, as in cells that express wild-type receptors, suggesting the presence of a kinase-regulated targeting signal in the cytoplasmic tail.
Expression and localisation of synaptotagmin isoforms in endocrine β-cells: their function in insulin exocytosis
Authors: Gut Anne , Kiraly Catherine Eva , Fukuda Mitsunori , Mikoshiba Katsuhiko , Wollheim Claes B. , Lang Jochen
Published: April 2021

Exocytosis of insulin containing Large Dense Core Vesicles (LDCVs) from pancreatic β-cells and derived cell lines is mainly controlled by Ca2+. Several lines of evidence have demonstrated a role of the Ca2+- and phospholipid-binding protein synaptotagmin (syt) in this event. Synaptotagmins form a large protein family with distinct affinities for Ca2+ determined by their two C2 domains (C2A/B). Except for the well-characterized isoforms I and II, their role is still unclear. We have used here insulin-secreting cells as a model system for LDCV exocytosis to gain insight into the function of synaptotagmins. Immunocytochemical analysis revealed that of the candidate Ca2+ sensors in LDCV exocytosis, syt III was not expressed in primary β-cells, whereas syt IV was only found adjacent to the TGN.

However, syt V-VIII isoforms were expressed at different levels in various insulin-secreting cells and in pancreatic islet preparations. In streptolysin-O permeabilized primary β-cells the introduction of recombinant peptides (100 nM) corresponding to the C2 domains of syt V, VII and VIII, but not of syt III, IV or VI, inhibited Ca2+-evoked insulin exocytosis by 30% without altering GTPγS-induced release. Our observations demonstrate that syt III and IV are not involved in the exocytosis of LDCVs from primary β-cells whereas V, VII and VIII may mediate Ca2+-regulation of exocytosis.
Nucleocytoplasmic transport in human astrocytes: decreased nuclear uptake of the HIV Rev shuttle protein
Authors: Neumann Markus , Afonina Elena , Ceccherini-Silberstein Francesca , Schlicht Stefan , Erfle Volker , Pavlakis George N. , Brack-Werner Ruth
Published: April 2021

Astrocytes are cellular targets for the human immunodeficiency virus (HIV) that limit virus production, owing, at least in part, to the diminished functionality of the viral post-transcriptional stimulatory factor Rev. To understand the trafficking process in astrocytes, we compared nucleocytoplasmic transport of Rev and various proteins with well-characterized nucleocytoplasmic transport features in human astrocytes and control cells (HeLa). Localization and trafficking characteristics of several cellular and viral proteins, as well as nuclear trafficking of classical peptide signals upon microinjection were similar in both cell types, indicating maintenance of general features of nucleocytoplasmic transport in astrocytes. Quantification of fluorescence in living cells expressing Rev fused to green fluorescent protein (GFP) indicated a strong shift in intracellular distribution of Rev in astrocytes, with 50-70% of Rev in the cytoplasm, whereas the cytoplasmic proportion of Rev in HeLa cells is around 10%. The dynamics of nucleocytoplasmic trafficking of Rev were compared in astrocytes and Rev-permissive cells by monitoring migration of Rev-GFP in cell fusions using highly sensitive time-lapse imaging. Nuclear uptake of Rev was dramatically retarded in homo-polykaryons of astrocytes compared with control cells. Diminished nuclear uptake of Rev was also observed in hetero-polykaryons of Rev-permissive cells and astrocytes. These results indicate that astrocytes contain a cytoplasmic activity that interferes with nuclear uptake of Rev. Our studies suggest a model in which Rev is prevented from functioning efficiently in astrocytes by specific alterations of its nucleocytoplasmic trafficking properties.

Movies available on-line: http://www.biologists.com/JCS/movies/jcs1709.html
Rabphilin dissociated from Rab3 promotes endocytosis through interaction with Rabaptin-5
Authors: Coppola Thierry , Hirling Harald , Perret-Menoud Véronique , Gattesco Sonia , Catsicas Stefan , Joberty Gérard , Macara Ian G. , Regazzi Romano
Published: April 2021

Rabphilin is a secretory vesicle protein that interacts with the GTP-bound form of the small GTPase Rab3. We investigated the involvement of Rabphilin in endocytosis using different point mutants of the protein. Overexpression of wild-type Rabphilin in the insulin-secreting cell line HIT-T15 did not affect receptor-mediated transferrin endocytosis. By contrast, Rabphilin V61A, a mutant that is unable to interact with Rab3, enhanced the rate of transferrin internalization. The effect of Rabphilin V61A was not mimicked by Rabphilin L83A, another mutant with impaired Rab3 binding. Careful analysis of the properties of the two mutants revealed that Rabphilin V61A and Rabphilin L83A are both targeted to secretory vesicles, have stimulatory activity on exocytosis, and bind equally well to α-actinin. However, Rabphilin L83A fails to interact with Rabaptin-5, an important component of the endocytotic machinery. These results indicate that Rabphilin promotes receptor-mediated endocytosis and that its action is negatively modulated by Rab3. We propose that the hydrolysis of GTP that is coupled to the exocytotic event disrupts the Rabphilin-Rab3 complex and permits the recruitment of Rabaptin-5 at the fusion site. Our data show that immediately after internalization the transferrin receptor and VAMP-2 colocalize on the same vesicular structures, suggesting that Rabphilin favors the rapid recycling of the components of the secretory vesicle.
Testase 1 (ADAM 24) a plasma membrane-anchored sperm protease implicated in sperm function during epididymal maturation or fertilization
Authors: Zhu Guo-Zhang , Myles Diana G. , Primakoff Paul
Published: April 2021

Plasma membrane-anchored proteases have key roles in cell signaling, migration and refashioning the cell surface and its surroundings. We report the first example of a plasma membrane-anchored protease on mature sperm, testase 1 (ADAM 24). Unlike other studied sperm ADAMs (fertilin α and β, cyritestin) whose metalloprotease domains are removed during sperm development, we found testase 1 retains an active metalloprotease domain, suggesting it acts as a protease on mature sperm. Testase 1 is a glycoprotein (molecular mass 88 kDa), localized to the equatorial region of the plasma membrane of cauda epididymal sperm. Typically, proteolytic removal of the pro-domain is an initial activation step for ADAM proteases. The pro-domain of the testase 1 precursor (108 kDa) is proteolytically removed as sperm transit the caput epididymis to produce processed (mature) testase 1 (88 kDa). Testase 1 is unique among all studied ADAMs in that its proteolytic processing occurs on the sperm plasma membrane instead of at an intracellular site (the Golgi). Using GST-fusion proteins and a synthetic testase 1 C-terminal peptide, we found that the cytoplasmic tail of testase 1 could be phosphorylated in vitro by protein kinase C (PKC). Thus testase 1 apparently has a cytoplasmic PKC phosphorylation site(s). Protein kinase C is known to stimulate other ADAMs’ protease activity. Because events of the acrosome reaction include PKC activation, we speculate that testase 1 protease function could be important in sperm penetration of the zona pellucida after sperm PKC is activated during the acrosome reaction.
Novel localization and possible functions of cyclin E in early sea urchin development
Authors: Schnackenberg Bradley J. , Marzluff William F.
Published: April 2021

In somatic cells, cyclin E-cdk2 activity oscillates during the cell cycle and is required for the regulation of the G1/S transition. Cyclin E and its associated kinase activity remain constant throughout early sea urchin embryogenesis, consistent with reports from studies using several other embryonic systems. Here we have expanded these studies and show that cyclin E rapidly and selectively enters the sperm head after fertilization and remains concentrated in the male pronucleus until pronuclear fusion, at which time it disperses throughout the zygotic nucleus. We also show that cyclin E is not concentrated at the centrosomes but is associated with condensed chromosomes throughout mitosis for at least the first four cell cycles. Isolated mitotic spindles are enriched for cyclin E and cdk2, which are localized to the chromosomes. The chromosomal cyclin E is associated with active kinase during mitosis. We propose that cyclin E may play a role in the remodeling of the sperm head and re-licensing of the paternal genome after fertilization. Furthermore, cyclin E does not need to be degraded or dissociated from the chromosomes during mitosis; instead, it may be required on chromosomes during mitosis to immediately initiate the next round of DNA replication.
dally, aDrosophilamember of the glypican family of integral membrane proteoglycans, affects cell cycle progression and morphogenesis via a Cyclin A-mediated process
Authors: Nakato Hiroshi , Fox Bethany , Selleck Scott B.
Published: April 2021

division abnormally delayed (dally) encodes an integral membrane proteoglycan of the glypican family that affects a number of patterning events during both embryonic and larval development. Earlier studies demonstrated that Dally regulates cellular responses to Wingless (Wg) and Decapentaplegic (Dpp) in a tissue-specific manner, consistent with its proposed role as a growth factor co-receptor. dally mutants also display cell cycle progression defects in specific sets of dividing cells in the developing optic lobe and retina. The affected cells in the retina and lamina show delays in completion of the G2-M segment of the cell cycle. We have investigated the molecular basis of dally-mediated cell division defects by examining the genetic interactions between dally and known cell cycle regulators.

Reductions in cyclin A but not cyclin B or string expression, suppress dally cell division defects in the optic lobe. cycA mutations also dominantly rescue many dally adult morphological defects including lethality, phenotypes that are unaffected by reducing cycB function. dally mutants show abnormal Cyclin A expression in the dividing cells affected, with appreciable levels of Cyclin A remaining in late prophase and metaphase, stages where Cyclin A is normally absent. Given that Dally is known to regulate the activity of secreted growth factors our findings suggest that extracellular cues influence the degradation of Cyclin A in a manner that controls cell cycle progression and ultimately, cell division patterning.
Altered mammary epithelial development, pattern formation and involution in transgenic mice expressing the EphB4 receptor tyrosine kinase
Authors: Munarini Nadia , Jäger Richard , Abderhalden Susanne , Zuercher Gisela , Rohrbach Valeria , Loercher Saemi , Pfanner-Meyer Brigitte , Andres Anne-Catherine , Ziemiecki Andrew
Published: April 2021

We have previously documented the cell-type-specific and hormone-dependent expression of the EphB4 receptor in the mouse mammary gland. To investigate its role in the biology of the mammary gland, we have established transgenic mice bearing the EphB4 receptor under the control of the MMTV-LTR promoter, which represents the first transgenic mouse model to investigate the effect(s) of unscheduled expression of EphB4 in adult organisms. Transgene expression in the mammary epithelium was induced at puberty, increased during pregnancy, culminated at early lactation and persisted until day three of post-lactational involution. In contrast, expression of the endogenous EphB4 gene is downregulated during pregnancy, is essentially absent during lactation and is re-induced after day three of post-lactational involution. The unscheduled expression of EphB4 led to a delayed development of the mammary epithelium at puberty and during pregnancy. During pregnancy, less lobules were formed, these however exhibited more numerous but smaller alveolar units. Transgenic mammary glands were characterized by a fragile, irregular morphology at lactation; however, sufficient functionality was maintained to nourish the young. Transgenic mammary glands exhibited untimely epithelial apoptotic cell death during pregnancy and abnormal epithelial DNA synthesis at early post-lactational involution, indicating a disturbed response to proliferative/apoptotic signals. Mammary tumours were not observed in the EphB4 transgenic animals; however, in double transgenic animals expressing both EphB4 and the neuT genes, tumour appearance was significantly accelerated and, in contrast to neuT-only animals, metastases were observed in the lung. These results implicate EphB4 in the regulation of tissue architecture, cellular growth response and establishment of the invasive phenotype in the adult mammary gland.
Integral membrane protein biosynthesis: why topology is hard to predict
Authors: Ott Carolyn M. , Lingappa Vishwanath R.
Published: April 2021

Integral membrane protein biogenesis requires the coordination of several events: accurate targeting of the nascent chain to the membrane; recognition,orientation and integration of transmembrane (TM) domains; and proper formation of tertiary and quaternary structure. Initially unanticipated inter-and intra-protein interactions probably mediate each stage of biogenesis for single spanning, polytopic and C-terminally anchored membrane proteins. The importance of these regulated interactions is illustrated by analysis of topology prediction algorithm failures. Misassigned or misoriented TM domains occur because the primary sequence and overall hydrophobicity of a single TM domain are not the only determinants of membrane integration.
The molecular basis for the autoregulation of calponin by isoform-specific C-terminal tail sequences
Authors: Burgstaller Gerald , Kranewitter Wolfgang J. , Gimona Mario
Published: April 2021

The three genetic isoforms of calponin (CaP), h1, h2 and acidic, are distinguished mostly by their individual C-terminal tail sequences. Deletion of these sequences beyond the last homologous residue Cys273 increases actin filament association for all three isoforms, indicating a negative regulatory role for the unique tail regions. We have tested this hypothesis by constructing a series of deletion and substitution mutants for all three CaP isoforms. Here we demonstrate that the C-terminal sequences regulate actin association by altering the function of the second actin-binding site, ABS2, in CaP comprised of the three 29-residue calponin repeats. Removal of the inhibitory tail resulted in an increased binding and bundling activity, and caused a prominent re-localization of h2 CaP from the peripheral actin network to the central actin stress fibers in transfected A7r5 smooth muscle cells. Domain-swap experiments demonstrated that the tail sequence of h2 CaP can downregulate cytoskeletal association efficiently in all three CaP isoforms, whereas the tail of the smooth-muscle-specific h1 CaP variant had little effect. Site-directed mutagenesis further revealed that the negatively charged residues within the tail region are essential for this regulatory function. Finally we demonstrate that the tail sequences regulate the second actin-binding site (ABS2) and not the strong actin-binding ABS1 region in CaP.
Integrin clustering induces kinectin accumulation
Authors: Tran Huan , Pankov Roumen , Tran Simon D. , Hampton Brian , Burgess Wilson H. , Yamada Kenneth M.
Published: April 2021

Integrin receptors mediate the formation of adhesion complexes and play important roles in signal transduction from the extracellular matrix. Integrin-based adhesion complexes (IAC) contain proteins that link integrins to the cytoskeleton and recruit signaling molecules, including vinculin,paxillin, focal adhesion kinase, talin and α-actinin. In this study, we describe a ∼160 kDa protein that is markedly enriched at IAC induced by clustering integrins with fibronectin-coated beads. Protein sequence analysis reveals that this ∼160 kDa protein is kinectin. Kinectin is an integral membrane protein found in endoplasmic reticulum, and it serves as a receptor for the motor protein kinesin. Fibronectin-induced IAC sequestered over half of the total cellular content of kinectin within 20 minutes. In addition, two other ER-resident proteins, RAP [low-density lipoprotein receptor-related protein (LRP) receptor-associated protein] and calreticulin, were found to be clustered at IAC, whereas kinesin was not. Our results identify a novel class of constituents of IAC.
Drac1 and Crumbs participate in amnioserosa morphogenesis during dorsal closure inDrosophila
Authors: Harden Nicholas , Ricos Michael , Yee Kelly , Sanny Justina , Langmann Caillin , Yu Hong , Chia William , Lim Louis
Published: April 2021

Dorsal closure of the Drosophila embryo involves morphological changes in two epithelia, the epidermis and the amnioserosa, and is a popular system for studying the regulation of epithelial morphogenesis. We previously implicated the small GTPase Drac1 in the assembly of an actomyosin contractile apparatus, contributing to cell shape change in the epidermis during dorsal closure. We now present evidence that Drac1 and Crumbs, a determinant of epithelial polarity, are involved in setting up an actomyosin contractile apparatus that drives amnioserosa morphogenesis by inducing apical cell constriction. Expression of constitutively active Drac1 causes excessive constriction of amnioserosa cells and contraction of the tissue, whereas expression of dominant-negative Drac1 impairs amnioserosa morphogenesis. These Drac1 transgenes may be acting through their effects on the amnioserosa cytoskeleton, as constitutively active Drac1 causes increased staining for F-actin and myosin, whereas dominant-negative Drac1 reduces F-actin levels. Overexpression of Crumbs causes premature cell constriction in the amnioserosa, and dorsal closure defects are seen in embryos homozygous for hypomorphic crumbs alleles. The ability of constitutively active Drac1 to cause contraction of the amnioserosa is impaired in a crumbsmutant background. We propose that amnioserosa morphogenesis is a useful system for studying the regulation of epithelial morphogenesis by Drac1.
Activation, processing and trafficking of extracellular heparanase by primary human fibroblasts
Authors: Nadav Liat , Eldor Amiram , Yacoby-Zeevi Oron , Zamir Eli , Pecker Iris , Ilan Neta , Geiger Benjamin , Vlodavsky Israel , Katz Ben-Zion
Published: April 2021

Heparanase is a heparan-sulfate-degrading endoglycosidase that has important roles in various biological processes, including angiogenesis, wound healing and metastatsis. Human heparanase is synthesized as a 65 kDa latent precursor, which is proteolytically processed into a highly active 50 kDa form. Extracellular heparanase is found in various tissues and is utilized by both normal cells and metastatic cancer cells to degrade heparan sulfate moieties in basement membranes and extracellular matrices. This study characterizes the processing and trafficking events associated with cellular activation of extracellular heparanase. We show that primary human fibroblasts are capable of binding and converting the 65 kDa heparanase precursor into its highly active 50 kDa form, concomitantly with its cytoplasmic accumulation. Heparanase uptake depends on the actin cytoskeleton integrity, resulting in a prolonged storage of the enzyme, mainly in endosomal structures. Heparanase endocytosis and its proteolytic activation are independent processes,indicating that heparanase cleavage is a cell surface event. Heparin completely inhibits heparanase endocytosis but only partially inhibits its association with the cells, suggesting that cell surface heparan sulfate moieties play a specific role in its endocytosis. Cellular binding and uptake of extracellular heparanase control its activation, clearance rate and storage within the cells.
Differential regulation of Rho GTPases by β1 and β3 integrins: the role of an extracellular domain of integrin in intracellular signaling
Authors: Miao Hui , Li Song , Hu Ying-Li , Yuan Suli , Zhao Yihua , Chen Benjamin P. C. , Puzon-McLaughlin Wilma , Tarui Takehiko , Shyy John Y.-J. , Takada Yoshikazu , Usami Shunichi , Chien Shu
Published: April 2021

Integrins mediate cell adhesion and signal transduction at focal adhesions. Here we investigate the roles of integrin β subunits in the regulation of actin cytoskeletal structure and the activities of Rho and Rac. The overexpression of β3 integrin in Chinese hamster ovary cells enhances Rho activity and stress fiber formation, whereas the overexpression of β1 integrin increases Rac activity and lamellipodia formation. The overexpression of a mutant β1-3-1 integrin, in which the extracellular I-domain-like sequence of β1 integrin has been replaced with the corresponding sequence of β3 integrin, also enhances Rho activity and the formation of stress fibers. Our results demonstrate that β1 and β3 integrins differentially regulate the activities of Rho family GTPases and that the extracellular domains of integrin β subunits play a critical role in transducing the extracellular ligand-binding information into specific intracellular signaling events.