bluuebunny/crossref_metadata_2025 huggingface dataset:
Asymmetric cell division: microtubule dynamics and spindle asymmetry
Authors: Kaltschmidt Julia A. , Brand Andrea H.
Published: April 2021

Asymmetric cell division can produce daughter cells with different developmental fates and is often accompanied by a difference in cell size. A number of recent genetic and in vivo imaging studies in Drosophilaand Caenorhabditis elegans have begun to elucidate the mechanisms underlying the rearrangements of the cytoskeleton that result in eccentrically positioned cleavage planes. As a result, we are starting to gain an insight into the complex nature of the signals controlling cytoskeletal dynamics in the dividing cell. In this commentary we discuss recent findings on how the mitotic spindle is positioned and on cleavage site induction and place them in the context of cell size asymmetry in different model organisms.
A ubiquitin C-terminal hydrolase is required to maintain osmotic balance and execute actin-dependent processes in the earlyC. elegansembryo
Authors: Kaitna Susanne , Schnabel Heinke , Schnabel Ralf , Hyman Anthony A. , Glotzer Michael
Published: April 2021

In the early Caenorhabditis elegans embryo, establishment of cell polarity and cytokinesis are both dependent upon reorganization of the actin cytoskeleton. Mutations in the cyk-3 gene cause maternal effect embryonic lethality. Embryos produced by homozygous cyk-3 mutant animals become multinucleate. We have further analyzed the cyk-3mutant phenotype and have found that cyk-3 mutant embryos fail to properly polarize the actin cytoskeleton and fail to segregate germline determinants. In addition, they fail to assemble an intact cleavage furrow. However, we have found that cyk-3 mutant embryos are intrinsically defective in osmotic regulation and that the cytokinesis defects can be partially rescued by providing osmotic support. The cyk-3 gene has been identified and found to encode a ubiquitin C-terminal hydrolase that is active against model substrates. These data indicate that the deubiquitination of certain substrates by CYK-3 is crucial for cellular osmoregulation. Defects in osmoregulation appear to indirectly affect actin-dependent processes.
CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains
Authors: Politi Valeria , Perini Giovanni , Trazzi Stefania , Pliss Artem , Raska Ivan , Earnshaw William C. , Valle Giuliano Della
Published: April 2021

CENP-C is a fundamental component of the centromere, highly conserved among species and necessary for the proper assembly of the kinetochore structure and for the metaphase-anaphase transition. Although CENP-C can bind DNA in vitro,the identification of the DNA sequences associated with it in vivo and the significance of such an interaction have been, until now, elusive. To address this problem we took advantage of a chromatin-immunoprecipitation procedure and applied this technique to human HeLa cells. Through this approach we could establish that: (1) CENP-C binds the alpha-satellite DNA selectively; (2) the CENP-C region between amino acids 410 and 537, previously supposed to contain a DNA-binding domain, is indeed required to perform such a function in vivo;and (3) the profile of the alpha-satellite DNA associated with CENP-C is essentially identical to that recognized by CENP-B. However, further biochemical and ultrastructural characterization of CENP-B/DNA and CENP-C/DNA complexes, relative to their DNA components and specific spatial distribution in interphase nuclei, surprisingly reveals that CENP-C and CENP-B associate with the same types of alpha-satellite arrays but in distinct non-overlapping centromere domains. Our results, besides extending previous observations on the role of CENP-C in the formation of active centromeres, show, for the first time, that CENP-C can associate with the centromeric DNA sequences in vivo and, together with CENP-B, defines a highly structured organization of the alpha-satellite DNA within the human centromere.
Intestinal stem cells protect their genome by selective segregation of template DNA strands
Authors: Potten Christopher S. , Owen Gary , Booth Dawn
Published: April 2021

The stem cells in the crypts of the small intestinal mucosa divide about a thousand times during the lifespan of a laboratory mouse, and yet they show little evidence of any decline in proliferative potential and rarely develop carcinogenic mutations, suggesting that their genome is extremely well protected. Protection against DNA-replication-induced errors can be achieved by the selective sorting of old (template) and new DNA strands with all template strands retained in the stem cell line. The template strands in the stem cells can be labelled during development or during tissue regeneration using tritiated thymidine (3HTdR). Labelling newly synthesised strands with a different marker (bromodeoxyuridine, BrdUrd) allows segregation of the two markers to be studied. Template strand label is retained(3HTdR), whereas label in the newly synthesised strands (BrdUrd) is lost following the second division of the stem cell. Random errors may occur in the template strands owing to environmental elements. These are protected against by the altruistic cell suicide (apoptosis) of the cells incurring such errors. A final level of protection for the tissue compensates for excessive deletion of stem cells via the apoptosis pathway. This is achieved by a hierarchical age structure in the stem cell compartment, with some cells being able to efficiently repair DNA damage and hence being more radioresistant. The presence of these protective mechanisms ensures that the small intestine rarely develops cancer and that stem cells can sustain the extensive cell proliferation needed during life.
BiP is feed-back regulated by control of protein translation efficiency
Authors: Gülow Karsten , Bienert Detlef , Haas Ingrid G.
Published: April 2021

The lumenal endoplasmic reticulum (ER) protein BiP, among its other functions, is believed to serve as an ER stress sensor, triggering the so-called `unfolded protein response' or UPR. For this role, BiP levels are critical. Indeed, here we show that BiP expression is tightly controlled at a post-transcriptional level. Thus, an artificial increase in cellular BiP mRNA does not lead to increased synthesis of BiP in unstressed cells, and,consequently, protein levels remain constant. Under ER stress conditions,however, this homeostatic restriction is alleviated, and independent of transcript levels, the translation efficiency of BiP transcripts is enhanced,allowing the cells to produce more protein. We additionally show that this regulation is independent of elements in the 5′ and 3′ UTR of BiP mRNA, which rather points to a novel type of translational feedback control. BiP is the first example of a lumenal protein whose expression is controlled at a translational level. The implications of these findings with respect to cellular stress are discussed.
Rho-dependent and -independent activation mechanisms of ezrin/radixin/moesin proteins: an essential role for polyphosphoinositides in vivo
Authors: Yonemura Shigenobu , Matsui Takeshi , Tsukita Shoichiro , Tsukita Sachiko
Published: April 2021

Ezrin/radixin/moesin (ERM) proteins crosslink actin filaments to plasma membranes and are involved in the organization of the cortical cytoskeleton,especially in the formation of microvilli. ERM proteins are reported to be activated as crosslinkers in a Rho-dependent manner and are stabilized when phosphorylated at their C-terminal threonine residue to create C-terminal threonine-phosphorylated ERM proteins (CPERMs). Using a CPERM-specific mAb, we have shown, in vivo, that treatment with C3 transferase (a Rho inactivator) or staurosporine (a protein kinase inhibitor) leads to the dephosphorylation of CPERMs, the translocation of ERM proteins from plasma membranes to the cytoplasm and microvillar breakdown. We further elucidated that ERM protein activation does not require C-terminal phosphorylation in A431 cells stimulated with epidermal growth factor. In certain types of kidney-derived cells such as MDCK cells, however, ERM proteins appear to be activated in the absence of Rho activation and remain active without C-terminal phosphorylation. Interestingly, microinjection of an aminoglycoside antibiotic, neomycin, which binds to polyphosphoinositides, such as phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2],affected the activation of ERM proteins regardless of cell type. These findings not only indicate the existence of a Rho-independent activation mechanism of ERM proteins but also suggest that both Rho-dependent and-independent activation of ERM proteins require a local elevation of PtdIns(4,5)P2 concentration in vivo.
Trimers of the fibronectin cell adhesion domain localize to actin filament bundles and undergo rearward translocation
Authors: Coussen Françoise , Choquet Daniel , Sheetz Michael P. , Erickson Harold P.
Published: April 2021

Previous studies have shown that small beads coated with FN7-10, a four-domain cell adhesion fragment of fibronectin, bind to cell surfaces and translocate rearward. Here we investigate whether soluble constructs containing two to five FN7-10 units might be sufficient for activity. We have produced a monomer, three forms of dimers, a trimer and a pentamer of FN7-10,on the end of spacer arms. These oligomers could bind small clusters of up to five integrins. Fluorescence microscopy showed that the trimer and pentamer bound strongly to the cell surface, and within 5 minutes were prominently localized to actin fiber bundles. Monomers and dimers showed only diffuse localization. Beads coated with a low concentration (probably one complex per bead) of trimer or pentamer showed prolonged binding and rearward translocation, presumably with the translocating actin cytskeleton. Beads containing monomer or dimer showed only brief binding and diffusive movements. We conclude that clusters of three integrin-binding ligands are necessary and sufficient for coupling to and translocating with the actin cytoskeleton.
Hyperproliferation, induction of c-Myc and 14-3-3σ, but no cell fragility in keratin-10-null mice
Authors: Reichelt Julia , Magin Thomas M.
Published: April 2021

In the past, keratins have been established as structural proteins. Indeed,mutations in keratin 10 (K10) and other epidermal keratins lead to severe skin fragility syndromes. Here, we present adult K10-/- mice, which reveal a novel connection between the regulation of cell proliferation and K10. Unlike most keratin mutant mice, the epidermis of adult K10-/-mice showed no cytolysis but displayed hyperproliferation of basal keratinocytes and an increased cell size. BrdU labelling revealed a shortened transition time for keratinocytes migrating outwards and DAPI staining of epidermal sheets uncovered an impaired organization of epidermal proliferation units. These remarkable changes were accompanied by the induction of c-Myc,cyclin D1, 14-3-3σ and of wound healing keratins K6 and K16. The phosphorylation of Rb remained unaltered. In line with the downregulation of K10 in squamous cell carcinomas and its absence in proliferating cells in vivo, our data suggest that the tissue-restricted expression of some members of the keratin gene family not only serves structural functions. Our results imply that the altered composition of the suprabasal cytoskeleton is able to alter the proliferation state of basal cells through the induction of c-Myc. A previous model based on transfection of K10 in immortalized human keratinocytes suggested a direct involvement of K10 in cell cycle control. While those experiments were performed in human cultured keratinocytes, our data establish, that in vivo, K10 acts by an indirect control mechanism in trans.
Cas, Fak and Pyk2 function in diverse signaling cascades to promote Yersinia uptake
Authors: Bruce-Staskal Pamela J. , Weidow Cheryl L. , Gibson Jennifer J. , Bouton Amy H.
Published: April 2021

The interplay between pathogen-encoded virulence factors and host cell signaling networks is critical for both the establishment and clearance of microbial infections. Yersinia uptake into host cells serves as an in vitro model for exploring how host cells respond to Yersinia adherence. In this study, we provide insight into the molecular nature and regulation of signaling networks that contribute to the uptake process. Using a reconstitution approach in Fak-/- fibroblasts, we have been able to specifically address the interplay between Fak, Cas and Pyk2 in this process. We show that both Fak and Cas play roles in the Yersinia uptake process and that Cas can function in a novel pathway that is independent of Fak. Fak-dependent Yersinia uptake does not appear to involve Cas-Crk signaling. By contrast, Cas-mediated uptake in the absence of Fak requires Crk as well as the protein tyrosine kinases Pyk2 and Src. In spite of these differences, the requirement for Rac1 activity is a common feature of both pathways. Furthermore, blocking the function of either Fak or Cas induces similar morphological defects in Yersinia internalization, which are manifested by incomplete membrane protrusive activity that is consistent with an inhibition of Rac1 activity. Pyk2 also functions in Yersinia uptake by macrophages, which are physiologically important for clearing Yersinia infections. Taken together, these data provide new insight into the host cellular signaling networks that are initiated upon infection with Y. pseudotuberculosis. Importantly, these findings also contribute to a better understanding of other cellular processes that involve actin remodeling, including the host response to other microbial pathogens, cell adhesion and migration.
Downregulated AP-1 activity is associated with inhibition of Protein-Kinase-C-dependent CD44 and ezrin localisation and upregulation of PKC theta in A431 cells
Authors: Stapleton Genevieve , Malliri Angeliki , Ozanne Bradford W.
Published: April 2021

Progression to an invasive, metastatic tumour requires the coordinated expression and function of a number of gene products, as well as their regulation in the context of invasion. The transcription factor AP-1 regulates expression of many of those genes necessary for implementation of the invasion programme. Two such gene products, CD44 and ezrin, are both upregulated in fibroblasts transformed by v-fos and are commonly implicated in cell motility and invasion. Here we report that CD44 and ezrin colocalise to membrane ruffles and microvilli of A431 cells after treatment with EGF. However, A431 cells expressing dominant-negative c-Jun (TAM67), and which as a consequence fail to invade in response to EGF, also fail to correctly localise CD44 and ezrin. CD44 and ezrin are both substrates for Protein Kinase C, and we show that their EGF-dependent colocalisation requires Protein Kinase C activity. Associated with TAM67 expression and disrupted CD44 and ezrin colocalisation is the increased expression and activation of the novel PKC theta isoform. Expression of PKC theta in A431 cells results in the inhibition of cell motility and disrupted localisation of CD44 and ezrin. We propose that AP-1 regulates the integrity of Protein Kinase C signalling and identifies PKC theta as a potential suppressor of the invasion programme.
Alterations in the lens capsule contribute to cataractogenesis in SPARC-null mice
Authors: Yan Qi , Clark John I. , Wight Thomas N. , Sage E. Helene
Published: April 2021

The lens capsule, which is also called the lens basement membrane, is a specialized extracellular matrix produced anteriorly by the lens epithelium and posteriorly by newly differentiated fiber cells. SPARC (secreted protein,acidic and rich in cysteine) is a matricellular glycoprotein that regulates cell-cell and cell-matrix interactions, cellular proliferation and differentiation, and the expression of genes encoding extracellular matrix components. SPARC-null mice exhibit lens opacity 1 month after birth and mature cataract and capsular rupture at 5-7 months. In this study, we report disruption of the structural integrity of the lens capsule in mice lacking SPARC. The major structural protein of basement membrane, collagen type IV, in the lens capsule was substantially altered in the absence of SPARC. The lens cells immediately beneath the capsule showed aberrant morphology, with numerous protrusions into the lens basement membrane. SPARC-null lenses at 1 month of age exhibited an increased penetration of dye or radioactive tracer through the capsule, as well as a higher content of water than their wild-type counterparts. Moreover, SPARC-null fibers exhibited swelling as early as 1 month of age; by 3 months, all the fiber cells appeared swollen to a marked degree. By contrast, the absence of SPARC had no apparent morphological effect on the early stages of lens formation, cell proliferation or fiber cell differentiation. Degradation of crystallins and MIP 26, or changes in the levels of these proteins, were not detected. These results underscore the importance of the capsular extracellular matrix in the maintenance of lens transparency and indicate that SPARC participates in the synthesis, assembly and/or stabilization of the lens basement membrane.
Regulation of S33/S37 phosphorylated β-catenin in normal and transformed cells
Authors: Sadot Einat , Conacci-Sorrell Maralice , Zhurinsky Jacob , Shnizer Dalia , Lando Zeev , Zharhary Dorit , Kam Zvi , Ben-Ze'ev Avri , Geiger Benjamin
Published: April 2021

A novel phosphorylation-specific antibody (αpβ-catenin) was generated against a peptide corresponding to amino acids 33-45 of humanβ-catenin, which contained phosphorylated serines at positions 33 and 37. This antibody is specific to phosphorylated β-catenin and reacts neither with the non-phosphorylated protein nor with phosphorylated or non-phosphorylated plakoglobin. It weakly interacts with S33Y β-catenin but not with the S37A mutant. pβ-catenin is hardly detectable in normal cultured cells and accumulates (up to 55% of total β-catenin) upon overexpression of the protein or after blocking its degradation by the proteasome. Inhibition of both GSK-3β and the proteasome resulted in a rapid (t1/2=10 minutes) and reversible reduction in pβ-catenin levels, suggesting that the protein can undergo dephosphorylation in live cells, at a rate comparable to its phosphorylation by GSK-3β. pβ-catenin interacts with LEF-1, but fails to form a ternary complex with DNA, suggesting that it is transcriptionally inactive. Immunofluorescence microscopy indicated that pβ-catenin accumulates in the nuclei of MDCK and BCAP cells when overexpressed and is transiently associated with adherens junctions shortly after their formation. pβ-catenin only weakly interacts with co-transfected N-cadherin, although it forms a complex with the ubiquitin ligase component β-TrCP. SW480 colon cancer cells that express a truncated APC, at position 1338, contain high levels of pβ-catenin,whereas HT29 cells, expressing APC truncated at position 1555, accumulate non-phosphorylated β-catenin, suggesting that the 1338-1555 amino acid region of APC is involved in the differential regulation of the dephosphorylation and degradation of pβ-catenin.
Ca2+-induced changes in SNAREs and synaptotagmin I correlate with triggered exocytosis from chromaffin cells: insights gleaned into the signal transduction using trypsin and botulinum toxins
Authors: Lawrence Gary W. , Dolly J. Oliver
Published: April 2021

Ca2+-triggered catecholamine exocytosis from chromaffin cells involves SNAP-25, synaptobrevin and syntaxin (known as SNAREs). Synaptotagmin I has been implicated as the Ca2+-sensor because it binds Ca2+, and this enhances its binding to syntaxin, SNAP-25 and phospholipids in vitro. However, most of these interactions are only mediated by [Ca2+]i two orders of magnitude higher than that needed to elicit secretion. Thus, the Ca2+ sensitivities of synaptotagmin I and the other SNAREs were quantified in situ. Secretion elicited from permeabilised cells by μM Ca2+ was accompanied,with almost identical Ca2+ dependencies, by changes in synaptotagmin I, SNAP-25, syntaxin and synaptobrevin that rendered them less susceptible to trypsin. The majority of the trypsin-resistant SNAREs were not associated with SDS-resistant complexes. None of these proteins acquired trypsin resistance in cells rendered incompetent for exocytosis by run-down. Removal of nine C-terminal residues from SNAP-25 by botulinum toxin A reduced both exocytosis and the SNAREs' acquisition of trypsin resistance but did not alter the Ca2+ sensitivity, except for synaptotagmin I. Even after synaptobrevin had been cleaved by botulinum toxin B, all the other proteins still responded to Ca2+. These data support a model whereby Ca2+ is sensed, probably by synaptotagmin I, and the signal passed to syntaxin and SNAP-25 before they interact with synaptobrevin.
Host cells: mobilizable lipid resources for the intracellular parasite Toxoplasma gondii
Authors: Charron Audra J. , Sibley L. David
Published: April 2021

Successful replication of the intracellular parasite Toxoplasma gondii within its parasitophorous vacuole necessitates a substantial increase in membrane mass. The possible diversion and metabolism of host cell lipids and lipid precursors by Toxoplasma was therefore investigated using radioisotopic and fluorophore-conjugated compounds. Confocal microscopic analyses demonstrated that Toxoplasma is selective with regards to both the acquisition and compartmentalization of host cell lipids. Lipids were compartmentalized into parasite endomembranes and, in some cases, were apparently integrated into the surrounding vacuolar membrane. Additionally,some labels became concentrated in discrete lipid bodies that were biochemically and morphologically distinct from the parasite apical secretory organelles. Thin layer chromatography established that parasites readily scavenged long-chain fatty acids as well as cholesterol, and in certain cases modified the host-derived lipids. When provided with radiolabeled phospholipid precursors, including polar head groups, phosphatidic acid and small fatty acids, intracellular parasites preferentially accrued phosphatidylcholine(PtdCho) over other phospholipids. Moreover, Toxoplasma was found to be competent to synthesize PtdCho from radiolabeled precursors obtained from its environment. Together, these studies underscore the ability of Toxoplasma gondii to divert and use lipid resources from its host, a process that may contribute to the biogenesis of parasite membranes.
Selective impairment of p53-mediated cell death in fibroblasts from sporadic Alzheimer's disease patients
Authors: Uberti Daniela , Carsana Teresina , Bernardi Enza , Rodella Luigi , Grigolato Piergiovanni , Lanni Cristina , Racchi Marco , Govoni Stefano , Memo Maurizio
Published: April 2021

In this study, we evaluated the response of different human skin fibroblast cultures obtained from eight probable Alzheimer's disease patients and eight non-Alzheimer's disease subjects to an acute oxidative injury elicited by H2O2. This treatment generates reactive oxygen species,which are responsible for DNA damage and apoptosis. To compare the sensitivity of fibroblasts from Alzheimer's disease or non-Alzheimer's disease patients to H2O2 exposure, we evaluated different parameters,including cell viability, the extension of DNA damage and the ability of the cells to arrest proliferation and to activate an apoptotic program. We found that fibroblasts from Alzheimer's disease patients were more resistant that those from control subjects to H2O2 treatment, although the extent of DNA damage induced by the oxidative injury was similar in both experimental groups. The protective mechanism of Alzheimer's disease fibroblasts was related to an impairment of H2O2-induced cell cycle arrest and characterized by an accelerated re-entry into the cell cycle and a diminished induction of apoptosis. Fibroblasts from Alzheimer's disease patients also have a profound impairment in the H2O2-activated, p53-dependent pathway, which results in a lack of activation of p53 or p53-target genes, including p21,GADD45 and bax. This study demonstrates a specific alteration of an intracellular pathway involved in sensing and repairing DNA damage in peripheral cells from Alzheimer's disease patients.
Rho5p downregulates the yeast cell integrity pathway
Authors: Schmitz Hans-Peter , Huppert Stefanie , Lorberg Anja , Heinisch Jürgen J.
Published: April 2021

The Rho family of proteins and their effectors are key regulators involved in many eukaryotic cell functions. In Saccharomyces cerevisiae the family consists of six members, Rho1p to Rho5p and Cdc42p. With the exception of Rho5p, these enzymes have been assigned different biological functions,including the regulation of polar growth, morphogenesis, actin cytoskeleton,budding and secretion. Here we show that a rho5 deletion results in an increased activity of the protein kinase C (Pkc1p)-dependent signal transduction pathway. Accordingly, the deletion shows an increased resistance to drugs such as caffeine, Calcofluor white and Congo red, which indicates activation of the pathway. In contrast, overexpression of an activated RHO5Q91H mutant renders cells more sensitive to these drugs. We conclude that Rho5p acts as an off-switch for the MAP-kinase cascade, which differentiates between MAP-kinase-dependent and -independent functions of Pkc1p. Kinetics of actin depolarisation and repolarisation after heat treatment of rho5 deletions as well as strains overexpressing the activated RHO5Q91H allele provide further evidence for such a function.
Occludin TM4-: an isoform of the tight junction protein present in primates lacking the fourth transmembrane domain
Authors: Ghassemifar M. Reza , Sheth Bhavwanti , Papenbrock Tom , Leese Henry J. , Houghton Franchesca D. , Fleming Tom P.
Published: April 2021

The tight junction protein occludin possesses four transmembrane domains,two extracellular loops, and cytoplasmic N- and C-termini. Reverse transcription-PCR analysis of human tissues, embryos and cells using primers spanning the fourth transmembrane domain (TM4) and adjacent C-terminal region revealed two products. The larger and predominant product corresponded in sequence to canonical occludin (TM4+), while the smaller product exhibited a 162 bp deletion encoding the entire TM4 and immediate C-terminal flanking region (TM4-). Examination of the genomic occludin sequence identified that the 162 bp sequence deleted in TM4-coincided precisely with occludin exon 4, strongly suggesting that TM4- is an alternative splice isoform generated by skipping of exon 4. Indeed, the reading frame of downstream exons is not affected by exclusion of exon 4. The presence of both TM4+ and TM4- occludin isoforms was also identified in monkey epithelial cells but TM4-was undetected in murine and canine tissue and cells, indicating a late evolutionary origin for this alternative splicing event. Conceptual translation of TM4- isoform predicts extracellular localisation of the C-terminus. Immunocytochemical processing of living human Caco-2 cells using a C-terminal occludin antibody revealed weak, discontinuous staining restricted to the periphery of subconfluent islands of cells, or islands generated by wounding confluent layers. In occludin immunoblots, a weak band at ∼58 kDa, smaller than the predominant band at 65 kDa and corresponding to the predicted mass of TM4- isoform, is evident and upregulated in subconfluent cells. These data suggest that the TM4- isoform may be translated at low levels in specific conditions and may contribute to regulation of occludin function.
InlB, a surface protein ofListeria monocytogenesthat behaves as an invasin and a growth factor
Authors: Bierne Hélène , Cossart Pascale
Published: April 2021

Molecules from some pathogenic bacteria mimic natural host cell ligands and trigger engulfment of the bacterium after specifically interacting with cell-surface receptors. The leucine-rich repeat (LRR)-containing protein InlB of Listeria monocytogenes is one such molecule. It triggers bacterial entry by interacting with the hepatocyte growth factor receptor (HGF-R or Met)and two other cellular components: gC1q-R and proteoglycans. Recent studies point to significant similarities between the molecular mechanisms underlying InlB-mediated entry into cells and classic phagocytosis. In addition, InlB, in common with HGF, activates signaling cascades that are not involved in bacterial entry. Therefore, studies of InlB may help us to analyze the previously noticed similarities between growth factor receptor activation and phagocytosis.
Regulation of focal complex composition and disassembly by the calcium-dependent protease calpain
Authors: Bhatt Amit , Kaverina Irina , Otey Carol , Huttenlocher Anna
Published: April 2021

Cell migration requires the regulated and dynamic turnover of adhesive complexes. We have previously demonstrated that the calcium-dependent protease, calpain, regulates the organization of adhesive complexes and cell detachment during cell migration. Evidence is now provided that inhibiting calpain through over-expression of the endogenous inhibitor of calpain,calpastatin, and pharmacological inhibitors results in an inhibition of adhesive complex disassembly with stabilization of GFP-vinculin and GFP/RFP-zyxin at the cell periphery. Calpain was also required for the microtubule-mediated turnover of adhesive complex sites after nocodazole wash-out, suggesting that calpain may mediate focal complex disassembly downstream of microtubules. Using dual imaging of RFP-zyxin and GFP—α-actinin, we observed a temporal and spatial relationship between α-actinin localization to focal contacts and the subsequent disassembly or translocation of RFP-zyxin containing focal complexes in areas of cell retraction. Calpain inhibition disrupted α-actinin localization to zyxin-containing focal contacts and focal complex disassembly or translocation to the cell center. In addition, disrupting α-actinin localization to focal complexes through expression of the α-actinin rod domain, but not the head domain, resulted in inhibition of focal adhesion disassembly similar to calpain inhibition. Our studies suggest a novel mechanism of action whereby calpain may modulate α-actinin localization into focal complexes and their subsequent disassembly or translocation.
Selective regulation of the Rab9-independent transport of ricin to the Golgi apparatus by calcium
Authors: Lauvrak Silje U. , Llorente Alicia , Iversen Tore-Geir , Sandvig Kirsten
Published: April 2021

Transport of ricin from endosomes to the Golgi apparatus occurs, in contrast to the transport of the mannose 6-phosphate receptor, by a Rab9-independent process. To characterize the pathway of ricin transport to the Golgi apparatus, we investigated whether it was regulated by calcium. As shown here, our data indicate that calcium is selectively involved in the regulation of ricin transport to the Golgi apparatus. Thapsigargin, which inhibits calcium transport into the ER, and the calcium ionophore A23187 both increased the transport of ricin to the Golgi apparatus by a factor of 20. By contrast, transport of the mannose 6-phosphate receptor to the Golgi apparatus was unaffected. Ricin and mannose 6-phosphate receptor transport were measured by quantifying the sulfation of modified forms of ricin and the mannose 6-phosphate receptor. The increased transport of ricin was reduced by wortmannin and LY294002, suggesting that phosphoinositide 3-kinase might be involved in transport of ricin to the Golgi apparatus. Together, these findings indicate that the different pathways to the Golgi apparatus utilized by ricin and the mannose 6-phosphate receptor are regulated by different mechanisms.
Active EGF receptors have limited access to PtdIns(4,5)P2 in endosomes: implications for phospholipase C and PI 3-kinase signaling
Authors: Haugh Jason M. , Meyer Tobias
Published: April 2021

Although prolonged cell signaling is attenuated by internalization and downregulation of active receptors, it is now appreciated that many receptors continue to signal in intracellular compartments. Employing enhanced green fluorescent protein fusion probes, we have investigated the hypothesis that multiple signaling pathways are affected by the differential trafficking of membrane substrates such as PtdIns(4,5)P2. A phosphotyrosine-specific probe, but not a PtdIns(4,5)P2-specific probe, colocalized with internalized EGF as well as transferrin in EGF-stimulated living cells expressing autophosphorylation-competent EGF receptors. Neither probe colocalized with transferrin in the absence of EGF, demonstrating that the reduced level of accessible PtdIns(4,5)P2 in endosomes is constitutive. Finally, a PtdIns(3,4,5)P3-specific probe, which monitors phosphorylation of PtdIns(4,5)P2 by phosphoinositide 3-kinases, was recruited to the plasma membrane but not to EGF- or transferrin-containing endosomes in response to EGF stimulation. These results suggest that while many internalized receptors continue to engage intracellular enzymes, the phospholipase C and phosphoinositide 3-kinase signaling pathways are abrogated by the constitutive lack of accessible PtdIns(4,5)P2 in endosomes.
Kinetics of HCMV immediate early mRNA expression in stably transfected fibroblasts
Authors: Snaar Sabine P. , Verdijk Pauline , Tanke Hans J. , Dirks Roeland W.
Published: April 2021

Compelling evidence supports an intimate link in time and space between eukaryotic pre-mRNA synthesis and processing and nucleocytoplasmic transport of mature mRNA. In this study, we analyzed the kinetic behavior of these processes in a quantitative manner. We used FISH and confocal scanning laser microscopy to detect transcripts produced by an inducible human cytomegalovirus immediate early (HCMV-IE) expression system. Upon induction, a large amount of pre-mRNA accumulated in nuclear foci at or near their transcription sites and, at later time, throughout the nucleoplasm. Inhibition of RNA polymerase II activity resulted in a rapid decrease in the number of transcripts in the nuclear RNA foci (half time ∼two minutes), indicating that accumulated transcripts were rapidly spliced and then released. The dispersed nucleoplasmic transcripts exited the nucleus with a half time of ∼10 minutes. Both processes were temperature dependent, suggesting that mRNA export is an active process. RNA polymerase II activation revealed that production of mature HCMV IE mRNAs required less than five minutes. Transcripts radiated from the gene at an average speed of ∼0.13 μm2/sec from this time on. Thus, it appears that these processes are tightly linked in time and space, with the splicing reaction as a rate-limiting factor.
Transient pluripotent cell populations during primitive ectoderm formation: correlation of in vivo and in vitro pluripotent cell development.
Authors: Pelton T. A. , Sharma S. , Schulz T. C. , Rathjen J. , Rathjen P. D.
Published: April 2021

Formation and differentiation of a pluripotent cell population is central to mammalian development, and the isolation, identification and manipulation of human pluripotent cells is predicted to be of therapeutic use. Within the early mammalian embryo, two distinct populations of pluripotent cells have been described: the inner cell mass (ICM), which differentiates to form a second pluripotent cell populations, the primitive ectoderm. Indirect evidence suggests the existence of temporally distinct intermediate pluripotent cell populations as primitive ectoderm is formed. We coupled an in vitro model of primitive ectoderm formation (the transition of embryonic stem cells to early primitive ectoderm-like (EPL) cells) with ddPCR-based techniques to identify three novel genes, Psc1, CRTR-1 and PRCE, that were expressed differently during pluripotent cell progression. Detailed mapping of these genes with Oct4, Rex1 and Fgf5 on pregastrulation embryos provided the first molecular evidence for the existence of successive, temporally distinct pluripotent cell populations in the embryo between the ICM and primitive ectoderm. No evidence was found for spatial heterogeneity within the Oct4+ pool. The transition between populations correlated with morphological or developmental alterations in pluripotent cells in vivo. Genes that are temporally expressed during pluripotent cell progression may provide an opportunity for molecular discrimination of pluripotent cells at different stages of maturation in vivo and an understanding of the cellular origins and properties of pluripotent cell lines isolated from diverse sources. Furthermore, the strong correlation of gene expression demonstrated between EPL cell formation in vitro and primitive ectoderm formation in vivo validates EPL cells as a model for primitive ectoderm, thereby providing a model system for the investigation of pluripotent differentiation and an opportunity for directed differentiation of pluripotent cells to therapeutically useful cell populations.
The scaffold protein IB1/JIP-1 controls the activation of JNK in rat stressed urothelium
Authors: Tawadros Thomas , Formenton Andrea , Dudler Jean , Thompson Nancy , Nicod Pascal , Leisinger Hans-Jürg , Waeber Gérard , Haefliger Jacques-Antoine
Published: April 2021

The c-Jun N-terminal kinase (JNK) is critical for cell survival, differentiation, apoptosis and tumorigenesis. This signalling pathway requires the presence of the scaffold protein Islet-Brain1/c-Jun N-terminal kinase interacting protein-1 (IB1/JIP-1). Immunolabeling and in situ hybridisation of bladder sections showed that IB1/JIP-1 is expressed in urothelial cells. The functional role of IB1/JIP-1 in the urothelium was therefore studied in vivo in a model of complete rat bladder outlet obstruction. This parietal stress, which is due to urine retention, reduced the content of IB1/JIP-1 in urothelial cells and consequently induced a drastic increase in JNK activity and AP-1 binding activity. Using a viral gene transfer approach, the stress-induced activation of JNK was prevented by overexpressing IB1/JIP-1. Conversely, the JNK activity was increased in urothelial cells where the IB1/JIP-1 content was experimentally reduced using an antisense RNA strategy. Furthermore, JNK activation was found to be increased in non-stressed urothelial cells of heterozygous mice carrying a selective disruption of the IB1/JIP-1 gene. These data established that mechanical stress in urothelial cells in vivo induces a robust JNK activation as a consequence of regulated expression of the scaffold protein IB1/JIP-1. This result highlights a critical role for that scaffold protein in the homeostasis of the urothelium and unravels a new potential target to regulate the JNK pathway in this tissue.
Multi-parameter analysis of the kinetics of NF-κB signalling and transcription in single living cells
Authors: Nelson Glyn , Paraoan Luminita , Spiller David G. , Wilde Geraint J. C. , Browne Mark A. , Djali Peter K. , Unitt John F. , Sullivan Elaine , Floettmann Eike , White Michael R. H.
Published: April 2021

Proteins of the NF-κB transcription factor family normally reside in the cytoplasm of cells in a complex with IκB inhibitor proteins. Stimulation with TNFα leads to proteosomal degradation of the IκB proteins and nuclear translocation of the NF-κB proteins. Expression of p65 and IκBα fused to fluorescent proteins was used to measure the dynamics of these processes in transfected HeLa cells. Simultaneous visualisation of p65-dsRed translocation and IκBα-EGFP degradation indicated that in the presence of dual fluorescent fusion protein expression,the half-time of IκBα-EGFP degradation was reduced and that of p65 translocation was significantly increased when compared with cells expressing the single fluorescent fusion proteins. These results suggest that the ratio of IκBα and p65 determine the kinetics of transcription factor translocation into the nucleus and indicate that the complex of p65 and IκBα is the true substrate for TNFα stimulation in mammalian cells.

When cells were treated with the CRM-1-dependent nuclear export inhibitor,leptomycin B (LMB), there was nuclear accumulation of IκBα-EGFP and p65-dsRed, with IκBα-EGFP accumulating more rapidly. No NF-κB-dependent transcriptional activation was seen in response to LMB treatment. Following 1 hour treatment with LMB, significant IκBα-EGFP nuclear accumulation, but low levels of p65-dsRed nuclear accumulation, was observed. When these cells were stimulated with TNFα, degradation of IκBα-EGFP was observed in both the cytoplasm and nucleus. A normal transient transcription response was observed in the same cells using luminescence imaging of NF-κB-dependent transcription. These observations suggest that both normal activation and post-induction repression of NF-κB-dependent transcription occur even when nuclear export of NF-κB is inhibited. The results provide functional evidence that other factors, such as modification of p65 by phosphorylation, or interaction with other proteins such as transcriptional co-activators/co-repressors, may critically modulate the kinetics of transcription through this signalling pathway.
Imaging of procollagen transport reveals COPI-dependent cargo sorting during ER-to-Golgi transport in mammalian cells
Authors: Stephens David J. , Pepperkok Rainer
Published: April 2021

We have examined the ER-to-Golgi transport of procollagen, which, when assembled in the lumen of the ER, is thought to be physically too large to fit in classically described 60-80 nm COPI- and COPII-coated transport vesicles. We found that procollagen exits the ER via COPII- coated ER exit sites and is transported to the Golgi along microtubules in defined transport complexes. These procollagen-containing transport complexes are, however, distinct from those containing other cargo proteins like ERGIC-53 and ts-045-G. Furthermore,they do not label for the COPI coat complex in contrast to those containing ts-045-G. Inhibition of COPII or COPI function before addition of ascorbate,which is required for the folding of procollagen, inhibits export of procollagen from the ER. Inactivation of COPI coat function after addition of ascorbate results in the localisation of procollagen to transport complexes that now also contain ERGIC-53 and are inhibited in their transport to the Golgi complex. These data reveal the existence of an early COPI-dependent,pre-Golgi cargo sorting step in mammalian cells.
Association of the tetraspanin CD151 with the laminin-binding integrinsα3β1, α6β1, α6β4 and α7β1 in cells in culture and in vivo
Authors: Sterk Lotus M. T. , Geuijen Cecile A. W. , van den Berg José G. , Claessen Nike , Weening Jan J. , Sonnenberg Arnoud
Published: April 2021

CD151 is a cell surface protein that belongs to the tetraspanin superfamily. It forms complexes with the laminin-binding integrinsα3β1, α6β1 and α6β4 and is codistributed with these integrins in many tissues at sites of cell-matrix interactions. In this study we show that CD151 can also form stable complexes with the laminin-binding integrin α7β1. The strength of this interaction is comparable to that between CD151 and α3β1. Complexes ofα3β1, α6β1 and α7β1 with CD151 are equally well formed with all splice variants of the α3, α6 and α7 subunits, and complex formation is not affected by mutations that prevent the cleavage of the integrin α6 subunit. Like the expression ofα3β1 and α6β1, expression of α7β1 in K562 cells results in increased levels of CD151 at its surface. Two non-integrin laminin receptors, dystroglycan and the polypeptide on which the Lutheran blood group antigens are expressed, are also often colocalized with CD151, but no association with CD151-α3β1 complexes was found with biochemical analysis.

The anti-CD151 antibody TS151R detects an epitope at a site at which CD151 interacts with integrins, and therefore it cannot react with CD151 when it is bound to an integrin. Comparison of the straining patterns produced by TS151R with that by of an anti-CD151 antibody recognizing an epitope outside the binding site (P48) revealed that most tissues expressing one or more laminin-binding integrins reacted with P48 but not with TS151R. However,smooth muscle cells that express α7β1 and renal tubular epithelial cells that express α6β1 were stained equally well by TS151R and P48. These results suggest that the interactions between CD151 and laminin-binding integrins are subject to cell-type-specific regulation.
Mitochondrial oxidative stress and cell death in astrocytes —requirement for stored Ca2+ and sustained opening of the permeability transition pore
Authors: Jacobson Jake , Duchen Michael R.
Published: April 2021

The role of oxidative stress is established in a range of pathologies. As mitochondria are a major source of reactive oxygen species (ROS), we have developed a model in which an intramitochondrial photosensitising agent is used to explore the consequences of mitochondrial ROS generation for mitochondrial function and cell fate in primary cells. We have found that, in astrocytes, the interplay between mitochondrial ROS and ER sequestered Ca2+ increased the frequency of transient mitochondrial depolarisations and caused mitochondrial Ca2+ loading from ER stores. The depolarisations were attributable to opening of the mitochondrial permeability transition pore (mPTP). Initially, transient events were seen in individual mitochondria, but ultimately, the mitochondrial potential(Δψm) collapsed completely and irreversibly in the whole population. Both ROS and ER Ca2+ were required to initiate these events, but neither alone was sufficient. Remarkably, the transient events alone appeared innocuous, and caused no increase in either apoptotic or necrotic cell death. By contrast, progression to complete collapse ofΔψ m caused necrotic cell death. Thus increased mitochondrial ROS generation initiates a destructive cycle involving Ca2+ release from stores and mitochondrial Ca2+-loading,which further increases ROS production. The amplification of oxidative stress and Ca2+ loading culminates in opening of the mPTP and necrotic cell death in primary brain cells.
Muscle regeneration by reconstitution with bone marrow or fetal liver cells from green fluorescent protein-gene transgenic mice
Authors: Fukada So-ichiro , Miyagoe-Suzuki Yuko , Tsukihara Hiroshi , Yuasa Katsutoshi , Higuchi Saito , Ono Shiro , Tsujikawa Kazutake , Takeda Shin'ichi , Yamamoto Hiroshi
Published: April 2021

The myogenic potential of bone marrow and fetal liver cells was examined using donor cells from green fluorescent protein (GFP)-gene transgenic mice transferred into chimeric mice. Lethally irradiated X-chromosome-linked muscular dystrophy (mdx) mice receiving bone marrow cells from the transgenic mice exhibited significant numbers of fluorescence+ and dystrophin+ muscle fibres. In order to compare the generating capacity of fetal liver cells with bone marrow cells in neonatal chimeras,these two cell types from the transgenic mice were injected into busulfantreated normal or mdx neonatal mice, and muscular generation in the chimeras was examined. Cardiotoxin-induced (or -uninduced, for mdx recipients) muscle regeneration in chimeras also produced fluorescence+ muscle fibres. The muscle reconstitution efficiency of the bone marrow cells was almost equal to that of fetal liver cells. However, the myogenic cell frequency was higher in fetal livers than in bone marrow. Among the neonatal chimeras of normal recipients, several fibres expressed the fluorescence in the cardiotoxin-untreated muscle. Moreover,fluorescence+ mononuclear cells were observed beneath the basal lamina of the cardiotoxin-untreated muscle of chimeras, a position where satellite cells are localizing. It was also found that mononuclear fluorescence+ and desmin+ cells were observed in the explantation cultures of untreated muscles of neonatal chimeras. The fluorescence+ muscle fibres were generated in the second recipient mice receiving muscle single cells from the cardiotoxin-untreated neonatal chimeras. The results suggest that both bone marrow and fetal liver cells may have the potential to differentiate into muscle satellite cells and participate in muscle regeneration after muscle damage as well as in physiological muscle generation.