bluuebunny/crossref_metadata_2025 huggingface dataset:
Ropporin, a sperm-specific binding protein of rhophilin, that is localized in the fibrous sheath of sperm flagella
Authors: Fujita Akiko , Nakamura Ken-ichi , Kato Takayuki , Watanabe Naoki , Ishizaki Toshimasa , Kimura Kazushi , Mizoguchi Akira , Narumiya Shuh
Published: April 2021

The small GTPase Rho functions as a molecular switch that regulates various cellular processes such as cell adhesion, motility, gene expression and cytokinesis. We previously isolated several putative Rho targets including rhophilin which bound selectively to the GTP-bound form of Rho. Rhophilin is expressed highly in testis and is localized specifically in sperm flagella. The presence of a PDZ domain at the carboxy terminus of rhophilin suggested that rhophilin works as an adaptor molecule. To test this hypothesis, we employed a yeast two hybrid system using the rhophilin PDZ domain as a bait, and screened a mouse testis cDNA library. We isolated several positive clones containing the same insert. The open reading frame of the cDNA encoded a novel protein of 212 amino acids designated as ropporin from a Japanese word ‘oppo’ (the tail). The amino-terminal 40 amino acid sequence of ropporin showed high homology to that of the regulatory subunit of type II cAMP-dependent protein kinase, which is involved in dimerization and binding to A-kinase anchoring proteins. Consistently, a yeast two hybrid assay and gel filtration of recombinant ropporin indicated that ropporin dimerizes through this domain. Deletion analysis indicated that the carboxy-terminal four amino acids are essential for binding of ropporin to rhophilin, and ropporin and RhoV14 coprecipitated in the presence of rhophilin in vitro. Northern blot analysis showed that ropporin is exclusively expressed in testis, and induced at the late stage of spermatogenesis. This induction paralleled that of rhophilin. Immunocytochemistry using anti-ropporin antibody showed that ropporin is localized in the principal piece and the end piece of sperm flagella. Electronmicroscopy revealed that ropporin is mostly localized in the inner surface of the fibrous sheath while rhophilin is present in the outer surface of the outer dense fiber. These results suggest that rhophilin and ropporin may form a complex in sperm flagella.
Yeast Golgi SNARE interactions are promiscuous
Authors: Tsui Marco M. K. , Banfield David K.
Published: April 2021

The transport of proteins between various compartments of the secretory pathway occurs by the budding of vesicles from one membrane and their fusion with another. A key event in this process is the selective recognition of the target membrane by the vesicle and the current view is that SNARE protein interactions likely play a central role in vesicle-target recognition and or membrane fusion. In yeast, only a single syntaxin (Sed5p) is required for Golgi transport and Sed5p is known to bind to at least 7 SNARE proteins. However, the number of Sed5p-containing SNARE complexes that exist in cells is not known. In this study we examined direct pair-wise interactions between full length soluble recombinant forms of SNAREs (Sed5p, Sft1p, Ykt6p, Vti1p, Gos1p, Sec22p, Bos1p, and Bet1p) involved in ER-Golgi and intra-Golgi membrane trafficking. In the binding assay that we describe here the majority of SNARE-binary interactions tested were positive, indicating that SNARE-SNARE interactions although promiscuous are not entirely non-selective. Interactions between a number of the genes encoding these SNAREs are consistent with our binding data and taken together our results suggest that functionally redundant Golgi SNARE-complexes exist in yeast. In particular, overexpression of Bet1p (a SNARE required for ER-Golgi and Golgi-ER traffic) and can bypass the requirement for the otherwise essential SNARE Sft1p (required for intra-Golgi traffic), suggesting that Bet1p either functions in a parallel pathway with Sft1p or can be incorporated into SNARE-complexes in place of Sftp1. None-the-less this result suggests that Bet1p can participate in two distinct trafficking steps, cycling between the ER and Golgi as well as in retrograde intra-Golgi traffic. In addition, suppressor genetics together with the analysis of the phenotypes of conditional mutations in Sft1p and Ykt6p, are consistent with a role for these SNAREs in more than one trafficking step.

We propose that different combinations of SNAREs form complexes with Sed5p and are required for multiple steps in ER-Golgi and intra-Golgi vesicular traffic. And that the apparent promiscuity of SNARE-SNARE binding interactions, together with the requirement for some SNAREs in more than one trafficking step, supports the view that the specificity of vesicle fusion events cannot be explained solely on the basis of SNARE-SNARE interactions.
Lymphoid adhesion promotes human thymic epithelial cell survival via NF-κB activation
Authors: Scupoli Maria T. , Fiorini Emma , Marchisio Pier C. , Poffe Ornella , Tagliabue Elda , Brentegani Monica , Tridente Giuseppe , Ramarli Dunia
Published: April 2021

Inside the thymus, thymic epithelial cells and thymocytes show an interdependent relationship for their functional differentiation and development. As regards possible interdependency for their mutual survival, it is clear that lympho-epithelial adhesion can control the survival of developing thymocytes whereas the effects of lymphoid adhesion on epithelial cell survival have never been described. To address this issue, we performed co-cultures between normal human thymic epithelial cells (TEC) and a mature lymphoid T cell line (H9) or unfractionated thymocytes. TEC were induced to apoptosis by growth factor deprivation and the level of cell death was measured by flow cytometry. TEC stimulated by cell adhesion showed a significant reduced apoptosis when compared to the control and this phenomenon was associated with increased binding activity of NF-κB, as measured by gel shift analysis. The activation of NF-κB was necessary to promote survival, since its inhibition by acetyl salicylic acid prevented the promoting effect. The mAb-mediated crosslinking of α3β1was considered as a potential inducer of TEC survival, since we have previously demonstrated that the engagement of this integrin was able to induce NF-κB activation in TEC. The crosslinking of α3β1, which clustered at the lympho-epithelial contact sites, partially reproduced the promoting activity of cell adhesion. These results highlight that lympho-epithelial adhesion can control the survival of thymic epithelial cells through an intracellular pathway which requires the activation of NF-κB and is triggered by integrins of the β1family.
HTLV-1-induced cell fusion is limited at two distinct steps in the fusion pathway after receptor binding
Authors: Daenke Susan , Booth Sarah
Published: April 2021

Human T-cell leukemia virus type 1 (HTLV-1) is notable among retroviruses for its poor ability to infect permissive cells, particularly as cell free virus. The virus is most efficiently transmitted between individuals by infected cells, where it is presumed that intracellular particles and viral RNA are transferred to target cells following fusion. Although the mandatory first step for HTLV-1 fusion is the binding of envelope SU (gp46) to the receptor, the events which follow this interaction and lead to fusion and infection have not been well characterized. To investigate these events, we studied two HTLV-1 chronically infected cell lines with different abilities to fuse with K562 target cells. Although not inherently fusion incompetent, the HTLV-1 envelope protein on MT2 cells was poorly able to undergo a change in membrane hydrophobicity required for fusion with the target cell membrane after binding to the receptor. High level expression of a fusion-competent HTLV-1 envelope protein on MT2 cells had little effect on improving this suggesting that the defect was encoded by the parent cell. Visible syncytia were seen after incubation of these cells with K562 target cells but complete fusion as measured by transfer of cellular contents into the recipient cell was not observed. In C91-PL cells, binding of SU to the receptor resulted in a sustained hydrophobic change of envelope accompanied by a cytopathic effect in mixed cell cultures and complete fusion. However, in C91-PL cells, overexpression of envelope protein blocked the transfer of cell contents after receptor engagement and initiation of cytopathic membrane changes, indicating that post binding fusion events were blocked. These data suggest that HTLV-1 fusion is a multistep process which is susceptible to inhibition at two seperate stages of the fusion pathway post receptor binding. This, and the inefficient infection by cellfree virions, may explain the poor infectivity of HTLV-1 in vivo and suggests strategies for preventative therapy.
Analysis of tight junctions during neutrophil transendothelial migration
Authors: Burns Alan R. , Bowden Robert A. , MacDonell Sonia D. , Walker David C. , Odebunmi Timothy O. , Donnachie Elizabeth M. , Simon Scott I. , Entman Mark L. , Smith C. Wayne
Published: April 2021

Intercellular junctions have long been considered the main sites through which adherent neutrophils (PMNs) penetrate the endothelium. Tight junctions (TJs; zonula occludens) are the most apical component of the intercellular cleft and they form circumferential belt-like regions of intimate contact between adjacent endothelial cells. Whether PMN transmigration involves disruption of the TJ complex is unknown. We report here that endothelial TJs appear to remain intact during PMN adhesion and transmigration. Human umbilical vein endothelial cell (HUVEC) monolayers, a commonly used model for studying leukocyte trafficking, were cultured in astrocyte-conditioned medium to enhance TJ expression. Immunofluorescence microscopy and immunoblot analysis showed that activated PMN adhesion to resting monolayers or PMN migration across interleukin-1-treated monolayers does not result in widespread proteolytic loss of TJ proteins (ZO-1, ZO-2, and occludin) from endothelial borders. Ultrastructurally, TJs appear intact during and immediately following PMN transendothelial migration. Similarly, transendothelial electrical resistance is unaffected by PMN adhesion and migration. Previously, we showed that TJs are inherently discontinuous at tricellular corners where the borders of three endothelial cells meet and PMNs migrate preferentially at tricellular corners. Collectively, these results suggest that PMN migration at tricellular corners preserves the barrier properties of the endothelium and does not involve widespread disruption of endothelial TJs.
Association of human ubiquitin-conjugating enzyme CDC34 with the mitotic spindle in anaphase
Authors: Reymond Françoise , Wirbelauer Christiane , Krek Wilhelm
Published: April 2021

Present in organisms ranging from yeast to man, homologues of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme CDC34 have been shown to play important roles in the regulation of cell cycle progression and checkpoint function. Here we analyze the expression and intracellular localization of endogenous CDC34 during mammalian cell cycle progression. We find that CDC34 protein is constitutively expressed during all stages of the cell cycle. Immunofluorescence experiments reveal that during interphase, endogenous CDC34 is localized to distinct speckles in both the nucleus and the cytoplasm. The presence of CDC34 in these compartments has also been established by biochemical fractionation experiments. Interestingly, nuclear localization depends on the presence of specific carboxy-terminal CDC34 sequences that have previously been shown to be required for CDC34’s cell cycle function in Saccharomyces cerevisiae. Finally, we find that in anaphase and not during early stages of mitosis, CDC34 colocalizes with β-tubulin at the mitotic spindle, implying that it may contribute to spindle function at later stages of mitosis. Taken together, these results support a model in which CDC34 ubiquitin-conjugating enzyme functions in the regulation of nuclear and cytoplasmic activities as well as in the process of chromosome segregation at the onset of anaphase in mammalian cells.
The S. pombe orthologue of the S. cerevisiae mob1 gene is essential and functions in signalling the onset of septum formation
Authors: Salimova Ekaterina , Sohrmann Marc , Fournier Nadine , Simanis Viesturs
Published: April 2021

We have isolated the Schizosaccharomyces pombe orthologue of the Saccharomyces cerevisiae MOB1 gene in a screen designed to enrich for septation mutants. The gene is essential, and cells lacking it display a phenotype typical of septation signalling network mutants. mob1p is located on both spindle pole bodies throughout mitosis. In addition it is also co-localised with the medial ring later in mitosis, and flanks the septum as the medial ring contracts. We also demonstrate that mob1p can be precipitated from cells in a complex with the septation regulating kinase sid2p.
Different effects of dominant negative mutants of desmocollin and desmoglein on the cell-cell adhesion of keratinocytes
Authors: Hanakawa Yasushi , Amagai Masayuki , Shirakata Yuji , Sayama Koji , Hashimoto Koji
Published: April 2021

Desmosomes contain two types of cadherin: desmocollin (Dsc) and desmoglein (Dsg). In this study, we examined the different roles that Dsc and Dsg play in the formation of desmosomes, by using dominant-negative mutants. We constructed recombinant adenoviruses (Ad) containing truncated mutants of E-cadherin, desmocollin 3a, and desmoglein 3 lacking a large part of their extracellular domains (EcadΔEC, Dsc3aΔEC, Dsg3ΔEC), using the Cre-loxP Ad system to circumvent the problem of the toxicity of the mutants to virus-producing cells. When Dsc3aΔEC Ad-infected HaCaT cells were cultured with high levels of calcium, E-cadherin and β-catenin, which are marker molecules for the adherens junction, disappeared from the cell-cell contact sites, and cell-cell adhesion was disrupted. This also occurred in the cells infected with EcadΔEC Ad. With Dsg3ΔEC Ad infection, keratin insertion at the cell-cell contact sites was inhibited and desmoplakin, a marker of desmosomes, was stained in perinuclear dots while the adherens junctions remained intact. Dsc3aΔEC Ad inhibited the induction of adherens junctions and the subsequent formation of desmosomes with the calcium shift, while Dsg3ΔEC Ad only inhibited the formation of desmosomes. To further determine whether Dsc3aΔEC directly affected adherens junctions, mouse fibroblast L cells transfected with E-cadherin (LEC5) were infected with these mutant Ads. Both Dsc3aΔEC and EcadΔEC inhibited the cell-cell adhesion of LEC5 cells, as determined by the cell aggregation assay, while Dsg3ΔEC did not. These results indicate that the dominant negative effects of Dsg3ΔEC were restricted to desmosomes, while those of Dsc3aΔEC were observed in both desmosomes and adherens junctions. Furthermore, the cytoplasmic domain of Dsc3aΔEC coprecipitated both plakoglobin and β-catenin in HaCaT cells. In addition, β-catenin was found to bind the endogenous Dsc in HaCaT cells. These findings lead us to speculate that Dsc interacts with components of the adherens junctions through β-catenin, and plays a role in nucleating desmosomes after the adherens junctions have been established.
Molecular mechanism of myosin-II assembly at the division site in Schizosaccharomyces pombe
Authors: Motegi Fumio , Nakano Kentaro , Mabuchi Issei
Published: April 2021

Schizosaccharomyces pombe cells divide by virtue of the F-actin-based contractile ring (F-actin ring). Two myosin-II heavy chains, Myo2 and Myp2/Myo3, have been localized to the F-actin ring. Here, we investigated the mechanism of myosin-II assembly at the division site in S. pombe cells.

First, we showed that Cdc4, an EF-hand protein, appears to be a common myosin light chain associated with both Myo2 and Myo3. Loss of function of both Myo2 and Myo3 caused a defect in F-actin assembly at the division site, like the phenotype of cdc4 null cells. It is suggested that Myo2, Myo3 and Cdc4 function in a cooperative manner in the formation of the F-actin ring during mitosis.

Next, we investigated the dynamics of myosin-II during mitosis in S. pombe cells. In early mitosis when accumulation of F-actin cables in the medial region was not yet observed, Myo2 was detected primarily as dots widely located in the medial cortex. Myo2 fibers also became visible following the appearance of the dots. The Myo2 dots and fibers then fused with each other to form a medial cortical network. Some Myo2 dots appeared to be localized with F-actin cables which are also accumulated in the medial region. Finally these structures were packed into a thin contractile ring. In mutant cells that cannot form the F-actin ring such as cdc3ts, cdc8ts and cdc12ts, Myo2 was able to accumulate as dots in the medial cortex, whereas no accumulation of Myo2 dots was detected in cdc4ts cells. Moreover, disruption of F-actin in the cell by applying latrunculin-A did not affect the accumulation of Myo2 dots, suggesting that F-actin is not required for their accumulation.

A truncated Myo2 which lacks putative Cdc4-binding sites (Myo2dIQs) was able to rescue myo2 null cells, myo3 null cells, cdc4ts mutant cells and cdc4 null cells. The Myo2dIQs could assemble into a normal-shaped ring in these cells. Therefore, its assembly at the division site does not require the function of either Cdc4 or Myo3.
Misdirected vimentin messenger RNA alters cell morphology and motility
Authors: Morris Eric J. , Evason Kim , Wiand Cheryl , L’Ecuyer Thomas J. , Fulton Alice B.
Published: April 2021

Localized messenger RNAs were first observed as embryonic determinants that altered development when mislocalized. In recent years localized mRNAs have been found for several cytoskeletal proteins, including actin, vimentin and several microtubule associated proteins. We sought to determine whether redirecting mRNA for a cytoskeletal protein to an inappropriate address would alter cellular phenotypes. To do so we generated vimentin mRNAs with a myc epitope tag and the β-actin 3′ untranslated region (3′ UTR) as a localization signal. When misdirected vimentin mRNAs are expressed in either fibroblasts or SW13 cells, cells develop numerous, extremely long processes; these cells also move more slowly to enter a wound of the monolayer. In situ hybridization revealed that the misdirected mRNA was often localized in the processes, in contrast to endogenous vimentin mRNA. The processes usually contained actin distal to the transgenic vimentin and microtubules proximal to it. SW13 cells lacking vimentin produced fewer and shorter processes, suggesting a dominant negative effect that involves recruitment of endogenous vimentin. Control experiments that transfected in constructs expressing tagged, correctly localized vimentin, or β-galactosidase that localized through the β-actin 3′ UTR, indicate that neither the shape nor the motility changes are solely due to the level of vimentin expression in the cell. This is direct evidence that the site of expression for at least one cytoskeletal mRNA alters the phenotype of the cell in which it is expressed. Messenger RNA localization is proving to be as essential for the normal maintenance of somatic cell phenotypes as embryonic determinants are for embryogenesis.
A role for TGFβ1 in osteoclast differentiation and survival
Authors: Fuller K. , Lean J. M. , Bayley K. E. , Wani M. R. , Chambers T. J.
Published: April 2021

Recently, tumour necrosis factorrelated activationinduced cytokine (TRANCE) was shown to be necessary for osteoclast formation. We now report that TGFβ, a cytokine enriched in bone matrix, is also required. TGFβ not only powerfully synergized with TRANCE for induction of osteoclastlike cells (OCL) from bone marrow precursors and monocytes, but OCL formation was abolished by recombinant soluble TGFβ receptor II (TGFβsRII). Preincubation in TGFβ was as effective as simultaneous incubation with TRANCE. TGFβ-preincubation enhanced OCL formation at least partly by preventing the development of resistance to OCL-induction that otherwise occurs when precursors are incubated in M-CSF. OCL formed in TRANCE also showed more rapid apoptosis than OCL in TRANCE plus TGFβ. Like TGFβ, incubation on bone matrix prolonged and enhanced the sensitivity of precursors to OCL-induction by TRANCE, and this was reversed by TGFβsRII. Taken together, this data is compelling evidence for a model in which TGFβ in matrix or released from bonelining or other cells maintains and enhances the osteoclastforming potential of precursors as they migrate towards sites of cellbound TRANCE. Thus, the specific circumstances necessary for osteoclast formation and survival are TRANCE expression on osteoblastic cells and TGFβ in bone.
A constitutive region is responsible for nuclear targeting of 4.1R: modulation by alternative sequences results in differential intracellular localization
Authors: Luque Carlos M. , Correas Isabel
Published: April 2021

Red blood cell protein 4.1, 4.1R, is an extreme variation on the theme of isoform multiplicity. The diverse 4.1R isoforms, mainly generated by alternative premRNA splicing, are localized at different intracellular sites, including the nucleus. To characterize nonerythroid 4.1 proteins lacking the most upstream translation initiation site, analyze their intracellular localization and define specific domains involved in differential intracellular targeting of 4.1R, we cloned 4.1 cDNAs lacking that translation initiation site.

Seven different 4.1R cDNAs were isolated. Four of these encoded 4.1R proteins localized predominantly to the nucleus and the other three localized to the cytoplasm. Three of the nuclear 4.1R isoforms did not contain the nuclear localization signal previously identified in the alternative exon 16. A comparative analysis of the exon composition of the naturally occurring 4.1R cDNAs cloned and of appropriate composite cDNA constructs, with the subcellular distribution of their respective products, demonstrated that a region encoded by constitutive exons, which is therefore common to all 4.1R isoforms and has been termed ‘core region’, had the capacity of localizing to the nucleus. This region was able to confer nuclear targeting to a cytosolic reporter. In protein 4.1R isoforms, the nuclear targeting of the core region is modulated by the expression of alternative exons. Thus, exon 5-encoded sequences eclipsed nuclear entry of the core region, resulting in 4.1R isoforms that predominantly distributed to the cytoplasm. Exon 5 was also able to confer cytoplasmic localization to a nuclear reporter. In protein 4.1R isoforms, when exons 5 and 16 were both expressed the nuclear targeting effect of exon 16 was dominant to the inhibitory effect observed by the expression of exon 5, yielding proteins that predominantly localized to the nucleus. Taken together, these results indicate that all 4.1R molecules contain a conserved region that is sufficient to target the protein to the nucleus, but that specific exonencoded sequences modulate this capacity by acting in a hierarchical order.
Role of the ribosome in sequence-specific regulation of membrane targeting and translocation of P-glycoprotein signal-anchor transmembrane segments
Authors: Zhang Jian-Ting , Han Ernest , Liu Yang
Published: April 2021

It is thought that the topology of a polytopic protein is generated by sequential translocation and membrane integration of independent signal-anchor and stop-transfer sequences. Two well-characterized cell-free systems (rabbit reticulocyte lysate and wheat germ extract) have been widely used to study the biogenesis of secretory and membrane proteins, but different results have been observed with proteins expressed in these two different systems. For example, different topologies of P-glycoprotein (Pgp) were observed in the two systems and the cause was thought to be the source of ribosomes. To understand how the ribosome is involved in dictating membrane translocation and orientation of polytopic proteins, individual signal-anchor sequences of Pgp were dissected and examined for their membrane targeting and translocation in a combined system of wheat germ ribosomes (WGR) and rabbit reticulocyte lysate (RRL). Addition of wheat germ ribosomes to the rabbit reticulocyte lysate translation system can enhance, reduce, or have no effect on the membrane targeting and translocation of individual Pgp signal-anchor sequences, and these effects appear to be determined by the amino acid residues flanking each signal-anchor. Ribosomes regulate the membrane targeting and translocation of Pgp signal-anchors in a polytopic form differently from the same signal-anchors in isolation. Furthermore, we demonstrated that ribosomes regulate the membrane targeting and translocation of each signal-anchor cotranslationally and that this activity of ribosomes is associated with the 60S subunit. Based on this and previous studies, we propose a mechanism by which ribosomes dynamically dictate the membrane targeting and translocation of nascent polytopic membrane proteins.
Syk-dependent phosphorylation of microtubules in activated B-lymphocytes
Authors: Faruki Shamsa , Geahlen Robert L. , Asai David J.
Published: April 2021

Syk is a protein-tyrosine kinase that is essential for B-lymphocyte development and B-cell signaling. Syk phosphorylates tubulin on tyrosine both in vitro and in intact lymphocytes. Here we show that α-tubulin present within the cytoskeletal microtubule network was phosphorylated in a Syk-dependent manner following the activation of B-cells by engagement of the B-cell antigen receptor or by treatment with the phosphotyrosine phosphatase inhibitor, pervanadate. Immunofluorescence staining of microtubule cytoskeletons and western blotting studies with antibodies to phosphotyrosine confirmed the phosphorylation of polymerized tubulin in Syk-expressing, but not Syk-deficient, cells. At low concentrations of pervanadate, centrosomes appeared to be preferentially tyrosine-phosphorylated. Tubulin phosphorylated to a high stoichiometry on tyrosine assembled into microtubules in vitro, and preassembled microtubules were also phosphorylated by Syk kinase in vitro. Thus, Syk has the capacity to interact with microtubule networks within the B-lymphocyte and catalyzes the phosphorylation of the α-tubulin subunit. Syk-dependent phosphorylation of microtubules may affect the ability of the microtubule cytoskeleton to serve as a platform upon which signaling complexes are assembled.
Identification and molecular-genetic characterization of a LAMP/CD68-like protein from Caenorhabditis elegans
Authors: Kostich Mitch , Fire Andrew , Fambrough Douglas M.
Published: April 2021

Lysosome associated membrane proteins (LAMPs) constitute a family of vertebrate proteins located predominantly in lysosomes, with lesser amounts present in endosomes and at the cell surface. Macrosialin/CD68s are similar to LAMPs in their subcellular distribution and amino acid sequence and presumed structure across the carboxyl terminal two thirds of their length. The functions of LAMPs and CD68s are not known. In the present study, a bioinformatics approach was used to identify a Caenorhabditis elegans protein (LMP-1) with sequence and presumed structural similarity to LAMPs and CD68s. LMP-1 appears to be the only membrane protein in C. elegans that carries a GYXXΦ vertebrate lysosomal targeting sequence at its C terminus (where Φ is a large, hydrophobic residue). LMP-1 was found to be present from early embryonic stages through adulthood and to be predominantly localized at the periphery of a population of large, membrane-bound organelles, called granules, that are seen throughout the early embryo but in later stages are restricted to the cells of the intestine. Analysis of an LMP-1 deficient C. elegans mutant revealed that LMP-1 is not required for viability under laboratory conditions, but the absence of LMP-1 leads to an alteration in intestinal granule populations, with apparent loss of one type of granule.
Different properties of two isoforms of annexin XIII in MDCK cells
Authors: Lecat Sandra , Verkade Paul , Thiele Christoph , Fiedler Klaus , Simons Kai , Lafont Frank
Published: April 2021

Annexins form a family of proteins that are widely expressed and known to bind membranes in the presence of calcium. Two isoforms of the annexin XIII subfamily are expressed in epithelia. We previously reported that annexin XIIIb is apically localized in MDCK cells and that it is involved in raft-mediated delivery of apical proteins. We have now analyzed the properties of annexin XIIIa, which differs from annexin XIIIb by a deletion of 41 amino acids in the amino-terminal domain, and is distributed both apically and basolaterally. Annexin XIIIa binding to membranes is independent of calcium but requires its myristoyl amino-terminal modification, as observed with annexin XIIIb. Our biochemical and functional data show that annexin XIIIa behaves differently in the apical and in the basolateral compartments. Whereas annexin XIIIa apically can associate with rafts independently of calcium, the basolateral pool requires calcium for this. Annexin XIIIa, like annexin XIIIb, stimulates apical transport of influenza virus hemagglutinin but, in contrast, only annexin XIIIa inhibits basolateral transport of vesicular stomatitis virus G protein. Our results suggest that annexin XIIIa and XIIIb have specific roles in epithelial cells, and because of their structural similarities, these isoforms offer interesting tools for unravelling the functions of annexins.
Sustained elevation in inositol 1,4,5-trisphosphate results in inhibition of phosphatidylinositol transfer protein activity and chronic depletion of the agonist-sensitive phosphoinositide pool
Authors: Speed Caroline J. , Mitchell Christina A.
Published: April 2021

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) in a signal-terminating reaction. We have utilised cell lines that stably underexpress the 43 kDa 5-phosphatase, as a model system to investigate whether Ins(1,4,5)P3 can control the rate of its own formation by regulating the resupply of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). A sustained 2.6-fold elevation in the basal concentration of Ins(1,4,5)P3, in cell lines underexpressing the 43 kDa 5-phosphatase, correlated with a 32% reduction in the total cellular mass of PtdIns(4,5)P2. The depletion in cellular PtdIns(4,5)P2 was confined to a Triton-insoluble cell compartment, enriched in caveolin. In resting cells with elevated Ins(1,4,5)P3 concentrations resulting from underexpression of the 43 kDa 5-phosphatase, phosphatidylinositol (PtdIns) and phosphatidylinositol 4-phosphate (PtdIns(4)P) were depleted by 50% and PtdIns(4,5)P2 by 61% in the caveolin-enriched Tritoninsoluble compartment. Agonist stimulation resulted in the rapid turnover of phosphoinositides in the caveolinenriched Triton-insoluble fraction of vector-transfected cells, but not in cells with high basal Ins(1,4,5)P3 concentrations. Depletion of phosphoinositides from the caveolin-enriched Triton-insoluble pool in cells underexpressing the 43 kDa 5-phosphatase did not result from activation of phospholipase C isoenzymes, or inhibition of PtdIns 4-kinase or PtdIns(4)P 5-kinase activities. Significant inhibition of phosphatidylinositol transfer protein (PITP) activity (up to 70%) was observed in cells with elevated basal Ins(1,4,5)P3 concentrations; however, no reduction in PITPα protein expression was detected. These studies indicate that chronic elevation in cellular Ins(1,4,5)P3 concentrations decreases the PITP-mediated resupply of phosphoinositides in the caveolinenriched agonist-sensitive pool.
Inhibiting proteasome activity causes overreplication of DNA and blocks entry into mitosis in sea urchin embryos
Authors: Kawahara Hiroyuki , Philipova Rada , Yokosawa Hideyoshi , Patel Rajnikant , Tanaka Keiji , Whitaker Michael
Published: April 2021

The proteasome has been shown to be involved in exit from mitosis by bringing about destruction of mitotic cyclins. Here, we present evidence that the proteasome is also required for proper completion of S phase and for entry into mitosis in the sea urchin embryonic cleavage cycle. A series of structurally related peptide-aldehydes prevent nuclear envelope breakdown in their order of inhibitory efficacies against the proteasome. Their efficacies in blocking exit from S phase and exit from mitosis correlate well, indicating that the proteasome is involved at both these steps. Mitotic histone HI kinase activation and tyrosine dephosphorylation of p34cdc2 kinase are blocked by inhibition of the proteasome, indicating that the proteasome plays an important role in the pathway that leads to embryonic p34cdc2 kinase activation. Arrested embryos continued to incorporate [3H]thymidine and characteristically developed large nuclei. Pre-mitotic arrest can be overcome by treatment with caffeine, a manoeuvre that is known to override the DNA replication checkpoint. These data demonstrate that the proteasome is involved in the control of termination of S phase and consequently in the initiation of M phase of the first embryonic cell cycle.
Differential distribution and defence involvement of antimicrobial peptides in mussel
Authors: Mitta Guillaume , Vandenbulcke Franck , Noël Thierry , Romestand Bernard , Beauvillain Jean Claude , Salzet Michel , Roch Philippe
Published: April 2021

In previous papers, we characterised 3 types of 4-kDa, cysteine-rich, cationic antimicrobial peptides: MGDs (for Mytilus galloprovincialis defensins), mytilins and myticins, which are abundant in the mussel hemocytes. In the present work, we revealed a differential distribution of the cells expressing the different genes. In addition, using confocal and electron microscopy, we confirmed that defensins and mytilins were partially located in different sub-types of circulating hemocytes although the peptides can be located in the same cell, and even in the same granule. We also demonstrated that mytilins exert their microbicidal effect within the cells through the process of phagosome-mytilin granule fusion leading to the co-location of ingested bacteria and mytilins.
The COOH-terminal nuclear localization sequence of interferon γ regulates STAT1α nuclear translocation at an intracellular site
Authors: Subramaniam Prem S. , Larkin Joseph , Mujtaba Mustafa G. , Walter Mark R. , Johnson Howard M.
Published: April 2021

We have recently shown that the nuclear localization of IFNγ is mediated by a polybasic nuclear localization sequence (NLS) in its C terminus. This NLS is required for the full expression of biological activity of IFNγ, both extracellularly and intracellularly. We now show that this NLS plays an integral intracellular role in the nuclear translocation of the transcription factor STAT1α activated by IFNγ. Treatment of IFNγ with antibodies to the C- terminal region (95-133) containing the NLS blocked the induction of STAT1α nuclear translocation. The antibodies had no effect on nuclear translocation of STAT1α in IFNα treated cells. A deletion mutant of human IFNγ, IFNγ(1-123), which is devoid of the C-terminal NLS region was found to be biologically inactive, but was still able to bind to the IFNγ receptor complex on cells with a Kd similar to that of the wild-type protein. Deletion of the NLS specifically abolished the ability of IFNγ(1-123) to initiate the nuclear translocation of STAT1α, which is required for the biological activities of IFNγ following binding to the IFNγ receptor complex. Thus, the NLS region appears to contribute minimally to extracellular high-affinity receptor-ligand binding, yet exerts a strong functional role in STAT1α nuclear localization. A high-affinity site for the interaction of the C-terminal NLS domain of IFNγ with a Kd approx. 3×10−8 M−1 has been described by previous studies on the intracellular cytoplasmic domain of the IFNγ receptor α-chain. To examine the role of the NLS at the intracellular level, we microinjected neutralizing antibodies raised against the C- terminal NLS domain of IFNγ into the cytoplasm of cells before treatment of cells with IFNγ. These intracellular antibodies specifically blocked the nuclear translocation of STAT1α following the subsequent treatment of these cells extracellularly with IFNγ. These data show that the NLS domain of IFNγ interacts at an intracellular site to regulate STAT1α nuclear import. A C-terminal peptide of murine IFNγ, IFNγ(95-133), that contains the NLS motif, induced nuclear translocation of STAT1α when taken up intracellularly by a murine macrophage cell line. Deletion of the NLS motif specifically abrogated the ability of this intracellular peptide to cause STAT1α nuclear translocation. In cells activated with IFNγ, IFNγ was found to as part of a complex that contained STAT1α and the importin-α analog Npi-1, which mediates STAT1α nuclear import. The tyrosine phosphorylation of STAT1α, the formation of the complex IFNγ/Npi-1/STAT1α complex and the subsequent nuclear translocation of STAT1α were all found to be dependent on the presence of the IFNγ NLS. Thus, the NLS of IFNγ functions intracellularly to directly regulate the activation and ultimate nuclear translocation STAT1α.
Actin disruption alters the localization of tau in the growth cones of cerebellar granule neurons
Authors: Zmuda Jonathan F. , Rivas Rodolfo J.
Published: April 2021

Cultured cerebellar granule neurons initially extend a single axon, followed by the extension of a second axon to attain a bipolar morphology. Differentiation culminates with the extension of several short dendrites from the cell body. In the present study, we determined the location of the dephosphorylated form of the microtubule-associated protein tau (dtau) within the growth cones of newly forming axons and examined whether this localization was influenced by the actin cytoskeleton. Following elongation of the initial axon at 2-3 days in vitro, dtau immunoreactivity was present along the entire length of the axon, becoming most intense just proximal to the growth cone. Dtau labeling dropped off dramatically along the microtubules of the growth cone and was undetectable along the most distal tips of these microtubules. As the initial axon continued to elongate at 3-4 days in vitro, the actin-rich growth cone peripheral domain characteristically underwent a dramatic reduction in size. Dtau immunoreactivity extended all the way to the most distal tips of the microtubules in the growth cones of these cells. Cytochalasin D and latrunculin A mimicked the effects of this characteristic reduction in growth cone size with regard to dtau localization in the growth cone. Depolymerization of filamentous actin caused the collapse of the peripheral domain and allowed dtau to bind all the way to the most distal tips of microtubules in the axon. Upon removal of the drugs, the peripheral domain of the growth cone rapidly re-formed and dtau was once again excluded from the most distal regions of growth cone microtubules. These findings suggest a novel role for actin in determining the localization of the microtubule- associated protein tau within the growth cones of neurons.
Human spectrin Src homology 3 domain binding protein 1 regulates macropinocytosis in NIH 3T3 cells
Authors: Xu Jiliu , Ziemnicka Dorota , Merz George S. , Kotula Leszek
Published: April 2021

Macropinocytosis is an endocytic process that occurs through non-clathrin coated vesicles larger than 0.2 μm in diameter. Although macropinocytic vesicles are readily visualized in cultured cells by the introduction of fluorescent, water-soluble dyes into the culture medium, protein markers associated with this type of vesicles have not yet been well defined. Here, we report that human spectrin SH3 domain binding protein 1, or Hssh3bp1, associates with macropinosomes in NIH 3T3 fibroblasts. Hssh3bp1 macropinosomes are heterogeneous in morphology and size, do not endocytose transferrin and are resistant to brefeldin A treatment. Cytochalasin D, and wortmannin block endocytosis of fluorescent dyes into the Hssh3bp1 macropinosomes and dramatically affect their morphology. Overexpression of Hssh3bp1-green fluorescent protein abolished fusion of vesicles resulting in a decreased endocytosis of fluorescence dyes, thus suggesting a potential regulatory role of Hssh3bp1 in macropinocytosis. In the macropinosomes of NIH 3T3 cells, Hssh3bp1 associates with a 200-kDa protein that crossreacts with a monoclonal antibody to the erythroid α-spectrin SH3 domain. Thus macropinosomes in cells may contain a spectrin-like protein.
The C. elegans septin genes, unc-59 and unc-61, are required for normal postembryonic cytokineses and morphogenesis but have no essential function in embryogenesis
Authors: Nguyen Tri Q. , Sawa Hitoshi , Okano Hideyuki , White John G.
Published: April 2021

Septins have been shown to play important roles in cytokinesis in diverse organisms ranging from yeast to mammals. In this study, we show that both the unc-59 and unc-61 loci encode Caenorhabditis elegans septins. Genomic database searches indicate that unc-59 and unc-61 are probably the only septin genes in the C. elegans genome. UNC-59 and UNC-61 localize to the leading edge of cleavage furrows and eventually reside at the midbody. Analysis of unc-59 and unc-61 mutants revealed that each septin requires the presence of the other for localization to the cytokinetic furrow. Surprisingly, unc-59 and unc-61 mutants generally have normal embryonic development; however, defects were observed in post-embryonic development affecting the morphogenesis of the vulva, male tail, gonad, and sensory neurons. These defects can be at least partially attributed to failures in post-embryonic cytokineses although our data also suggest other possible roles for septins. unc-59 and unc-61 double mutants show similar defects to each of the single mutants.
Single granule pH cycling in antigen-induced mast cell secretion
Authors: Williams Rebecca M. , Webb Watt W.
Published: April 2021

The pH cycling of individual granules in secreting (serotonin-loaded) mast cells is quantitatively examined using multicolor multiphoton fluorescence microscopy. A typical exocytosis event consists of maximal calcium rise at time zero, granule alkalization a few seconds later and, finally, complete contents release at a fraction of a second after alkalization. Membrane fusion is either transient, as indicated by subsequent granule reacidification, or ‘full’, as indicated by a granule disappearance with a collapse of its membrane into the plasma membrane. The relative frequency of these two coexisting behaviors (the ‘kiss-to-collapse’ ratio) is approximately 2:1. A typical transiently fusing granule experiences multiple alkalization/ acidification cycles after addition of exogenous antigen. Between recycling granules, coalescence events are frequent, with 80% resulting in a collapse of the formed granule complex to the plasma membrane. The full dynamics of secretion encompass a complex combination of these granule activities.

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Chk1 and Cds1: linchpins of the DNA damage and replication checkpoint pathways
Authors: Rhind Nicholas , Russell Paul
Published: April 2021

Recent work on the mechanisms of DNA damage and replication cell cycle checkpoints has revealed great similarity between the checkpoint pathways of organisms as diverse as yeasts, flies and humans. However, there are differences in the ways these organisms regulate their cell cycles. To connect the conserved checkpoint pathways with various cell cycle targets requires an adaptable link that can target different cell cycle components in different organisms. The Chk1 and Cds1 protein kinases, downstream effectors in the checkpoint pathways, seem to play just such roles. Perhaps more surprisingly, the two kinases not only have different targets in different organisms but also seem to respond to different signals in different organisms. So, whereas in fission yeast Chk1 is required for the DNA damage checkpoint and Cds1 is specifically involved in the replication checkpoint, their roles seem to be shuffled in metazoans.
Active protein transport through plastid tubules: velocity quantified by fluorescence correlation spectroscopy
Authors: Köhler Rainer H. , Schwille Petra , Webb Watt W. , Hanson Maureen R.
Published: April 2021

Dynamic tubular projections emanate from plastids in certain cells of vascular plants and are especially prevalent in non-photosynthetic cells. Tubules sometimes connect two or more different plastids and can extend over long distances within a cell, observations that suggest that the tubules may function in distribution of molecules within, to and from plastids. In a new application of two-photon excitation (2PE) fluorescence correlation spectroscopy (FCS), we separated diffusion of fluorescent molecules from active transport in vivo. We quantified the velocities of diffusion versus active transport of green fluorescent protein (GFP) within plastid tubules and in the cytosol in vivo. GFP moves by 3-dimensional (3-D) diffusion both in the cytosol and plastid tubules, but diffusion in tubules is about 50 times and 100 times slower than in the cytosol and an aqueous solution, respectively. Unexpectedly larger GFP units within plastid tubules exhibited active transport with a velocity of about 0.12 μm/second. Active transport might play an important role in the long-distance distribution of large numbers of molecules within the highly viscous stroma of plastid tubules.
Analysis of targeting sequences demonstrates that trafficking to the Toxoplasma gondii plastid branches off the secretory system
Authors: DeRocher Amy , Hagen Christopher B. , Froehlich John E. , Feagin Jean E. , Parsons Marilyn
Published: April 2021

Apicomplexan parasites possess a plastid-like organelle called the apicoplast. Most proteins in the Toxoplasma gondii apicoplast are encoded in the nucleus and imported post-translationally. T. gondii apicoplast proteins often have a long N-terminal extension that directs the protein to the apicoplast. It can be modeled as a bipartite targeting sequence that contains a signal sequence and a plastid transit peptide. We identified two nuclearly encoded predicted plastid proteins and made fusions with green fluorescent protein to study protein domains required for apicoplast targeting. The N-terminal 42 amino acids of the apicoplast ribosomal protein S9 directs secretion of green fluorescent protein, indicating that targeting to the apicoplast proceeds through the secretory system. Large sections of the S9 predicted transit sequence can be deleted with no apparent impact on the ability to direct green fluorescent protein to the apicoplast. The predicted transit peptide domain of the S9 targeting sequence directs protein to the mitochondrion in vivo. The transit peptide can also direct import of green fluorescent protein into chloroplasts in vitro. These data substantiate the model that protein targeting to the apicoplast involves two distinct mechanisms: the first involving the secretory system and the second sharing features with typical chloroplast protein import.