bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of the vimentin intermediate filament system during adipose conversion of 3T3-L1 cells inhibits lipid droplet accumulation
Authors: Lieber Jonathan G. , Evans Robert M.
Published: April 2021

During the differentiation of 3T3-L1 pre-adipocytes, vimentin intermediate filaments are reorganized to form cage-like structures around the nascent lipid droplets. Initial studies with 3T3-L1 cells indicated that aggregation of vimentin filaments by nocodazole treatment during or shortly after induction of adipose conversion dramatically reduced the lipid droplet content of 3T3-L1 cells 96-120 hours after induction. Specific but transient disruption of vimentin following anti-IFA antibody injection also resulted in a decrease in lipid droplet formation in differentiating cells. To specifically and stably affect filament organization, 3T3-L1 cells lines were established by transfection with a glucocorticoid-regulatable, dominant negative mutant vimentin cDNA expression plasmid. Treatment of these cells (83∆C) with dexamethasone resulted in expression of vimentin with a carboxyl-terminal deletion, which led to the disruption of the endogenous filament network. Induction of adipose conversion in 83∆C cells lead to the formation of lipid droplets comparable to those seen in untransfected 3T3-L1 cells. Addition of dexamethasone during the adipose conversion of 83∆C cells did not affect the induction of the marker enzyme glycerol-3-phosphate dehydrogenase or the incorporation of [14C]palmitate into triglycerides during a 10 minute pulse label. There was, however, a failure to form prominent lipid droplets and to accumulate [14C]palmitate-labeled triglycerides. Pulse-chase experiments indicated that the failure of these cells to accumulate triglyceride was associated with an increased rate of turnover. These studies indicate that vimentin filaments provide a function that influences lipid stability during adipose conversion of 3T3-L1 cells.
Cellulose microfibrils, cell motility, and plasma membrane protein organization change in parallel during culmination in Dictyostelium discoideum
Authors: Grimson Mark J. , Haigler Candace H. , Blanton Richard L.
Published: April 2021

Prestalk cells of Dictyostelium discoideum contribute cellulose to two distinct structures, the stalk tube and the stalk cell wall, during culmination. This paper demonstrates by freeze fracture electron microscopy that two distinct types of intramembrane particle aggregates, which can be characterized as cellulose microfibril terminal complexes, occur in the plasma membranes of cells synthesizing these different forms of cellulose. The same terminal complexes were observed in situ in developing culminants and in vitro in monolayer cells induced to synthesize the two types of cellulose. We propose that cessation of cell motility is associated with a change in packing and intramembrane mobility of the particle aggregates, which causes a change in the nature of the cellulose synthesized. The terminal complexes are compared to those described in other organisms and related to the previous hypothesis of two modes of cellulose synthesis in Dictyostelium.
A novel structural component of the Dictyostelium centrosome
Authors: Kalt Astrid , Schliwa Manfred
Published: April 2021

The microtubule-organizing center of D. discoideum is a nucleus-associated body (NAB) that consists of a multilayered, box-shaped core embedded in an amorphous corona from which the microtubules emerge. The composition of the NAB is still largely unresolved. Here we have examined a high molecular mass component of the NAB which was identified by a monoclonal antibody raised against isolated nucleus/NAB complexes. This antibody recognized a 350 kDa component which is immunologically related to the D. discoideum heavy chain of myosin II. The 350 kDa antigen was localized only at the NAB in interphase cells, while in mitotic cells it may also be found in the vicinity of the NAB as well as in association with the mitotic spindle. Immunogold labeling experiments showed that the protein is part of the NAB corona. This association was not destroyed by treatment with 2 M urea or 0.6 M KCl. The 350 kDa antigen was part of the thiabendazole-induced cytoplasmic microtubule-organizing centers. A direct role in the polymerization of tubulin could not be determined in an in vitro microtubule nucleation assay, whereas antibody electroporation of live cells appeared to interfere with the generation of a normal microtubule system in a subset of cells. Our observations suggest that the 350 kDa antigen is a structural component of the NAB corona which could be involved in its stabilization.
Dynamic changes of NuMA during the cell cycle and possible appearance of a truncated form of NuMA during apoptosis
Authors: Hsu Hsin-Ling , Yeh Ning-Hsing
Published: April 2021

We have demonstrated that dynamic redistribution of nuclear-mitotic apparatus (NuMA) protein in the cell cycle is correlated temporally and spatially with its biochemical modifications. In interphase, NuMA behaves solely as a 220 kDa nuclear matrix-associated protein. After initiation of DNA condensation during mitosis, NuMA is phosphorylated by Cdc2 kinase into a 240 kDa form which is transported quickly to the centrosomal region. Once cells have passed the metaphase-anaphase transition, the 240 kDa form of NuMA either becomes a 180 kDa truncated form which is fated to be degraded completely before mitotic exit, or returns to the 220 kDa form that relocates to the daughter nuclei and remains throughout interphase. Apparently, a proteolytic enzyme is activated during the late stages of mitosis. After induction of a 180 kDa form of NuMA in interphase HeLa cells by 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, nuclear apoptotic phenomena including chromatin condensation, DNA fragmentation, and micronucleation were observed. However, the same treatment did not induce apoptosis in mitotic phasearrested HeLa cells. The 180 kDa form of NuMA was demonstrated to be a truncated product, at least lacking the tail domain. When HL60 cells were stimulated by diverse apoptosis inducers such as camptothecin, staurosporine, cycloheximide, and A23187, the extent of NuMA cleavage to produce a 180 kDa product was comparable with the degree of oligonucleosomal laddering. NuMA cleavage is likely to be a consequence of the onset of apoptosis. The intact 220 kDa NuMA functions in interphase cells to retain the nuclear structural integrity. Additionally, NuMA appears to act as a nuclear structural target for a death protease during apoptosis.
Conditional differentiation of heartand smooth muscle-derived cells transformed by a temperature-sensitive mutant of SV40 T antigen
Authors: Jahn Lothar , Sadoshima Jun-ichi , Greene Adam , Parker Chris , Morgan Kathleen G. , Izumo Seigo
Published: April 2021

To create muscle cell lines that conditionally differentiate in vitro we introduced a temperature-sensitive SV40 T antigen by retroviral infection into rat aortic smooth muscle cells (SMCs) and neonatal heart-derived cells. After G418 selection cell lines isolated were characterized at permissive (33°C) and non-permissive (39°C) temperatures. [3H]Thymidine uptake showed that progression through the cell cycle is greatly reduced at 39°C. Cytoskeletal proteins, such as actins and vimentin did not change significantly after temperature shift, while the number of desmin-positive SMCs significantly increased when cells were switched to 39°C. Heart-derived muscle cells showed sarcomeric myosin heavy chain reactivity only when grown at 39°C. After thrombin stimulation intracellular calcium in both cell types increased severalfold in 39°C-cells but not in 33°C-cells. Whole cell patch-clamp recordings of SMCs and heart-derived cells revealed a strong increase in nicardipine-sensitive Ca2+ current when cells were switched to 39°C. Nicardipine-insensitive Ca2+ current also increased in both cell types at the non-permissive temperature. Na+ current in SMCs was large at 33°C and small or not detectable at 39°C and absent in heart-derived cells. Using a cDNA probe specific for the α1 subunit of the dihydropyridine-sensitive Ca2+ channel we demonstrate a temperature-sensitive expression of the dihydropyridine receptor mRNA in smooth muscle-derived cells but not in heart-derived H10 cells. Our results suggest that upon downregulation of SV40 T antigen these cells become quiescent and exhibit a more differentiated phenotype. These cell lines may provide a useful tool to investigate ion channeland receptor signal transduction, as well as cell cycle control in smooth and possibly cardiac muscle cell differentiation.
Hysteretic behavior and differential apparent stability properties of microtubule species emerge from the regulation of post-translational modifications of microtubules
Authors: Laurent Michel , Fleury Anne
Published: April 2021

At the epigenetic level, microtubule diversity is generated by several mechanisms of reversible post-translational modifications of tubulin subunits. In most cases, modification enzymes preferentially act on the tubulin subunits of microtubules, whereas the substrate of the enzymes which ensure the reverse reaction is preferentially the αβ-dimer of nonpolymerized tubulin. Most modifications identified to date appear to be nearly ubiquitous within the animal kingdom. Moreover, modifications are generally not mutually exclusive, so that cellular microtubules often bear several distinct biochemical alterations whose biological role is yet unknown.

Post-translational modifications often (but not always) occur on microtubule species with low turnover rate. However, in vitro comparison of the polymerization and depolymerization rates of modified or unmodified forms of tubulin did not reveal any significant difference between molecular species. Thus, post-translational modifications are thought to be the result rather than the cause of microtubule stability.

We re-examine this contention in the light of a regulated kinetic scheme for multiple and non-exclusive enzymatic modifications of microtubules. This study shows that different apparent stability properties of microtubule species emerge under such a kinetic regulation, although all the species were assumed to have identical intrinsic stability properties. This model can be used to reinterpret the results of well-known studies bearing on the relationship between microtubule stability and post-translational modifications. Another important finding is that the existence of a regulation loop in one of the multiple pathways of enzymatic differentiation of microtubules endows the system with hysteretic properties. These properties may be viewed, under restrictive conditions, as a buffering mechanism for the transitions between microtubule growing and shrinking phases during fluctuations in the regulation of centrosomal nucleating activity.
Identification of αvβ3 as a heterotypic ligand for CD31/PECAM-1
Authors: Buckley C. D. , Doyonnas R. , Newton J. P. , Blystone S. D. , Brown E. J. , Watt S. M. , Simmons D. L.
Published: April 2021

CD31 (PECAM-1) is a member of the immunoglobulin gene superfamily (IgSF) and has an important role in a number of endothelial cell functions including angiogenesis, inflammation, integrin activation and cell-cell adhesion. CD31 has both homotypic and heterotypic adhesive properties and in common with other IgSF members contains multiple functional domains. Using chimaeric fusion proteins of CD31 and a panel of haematopoietic cell lines we show that CD31 can bind cells in a predominantly homotypic or heterotypic manner depending on the cell line used. Heterotypic binding was found to be cation and temperature dependent and enhanced by Mn2+: all features of integrin mediated binding. Using a panel of anti-CD31 and anti-integrin antibodies we show that αvβ3 is a ligand for CD31 on the monocytic cell line U937. The specificity of the interaction between αvβ3 and CD31 was further confirmed by solid phase binding assays and the use of αvβ3 transfected cells which bound CD31 specifically. Furthermore, we have mapped the binding site for αvβ3 to domains 1 and 2 of CD31. The interaction of CD31 with αvβ3 may be important in many aspects of endothelial function including leukocyteendothelial transmigration and angiogenesis.
Contributions of the structural domains of filensin in polymer formation and filament distribution
Authors: Goulielmos George , Remington Susann , Schwesinger Falk , Georgatos Spyros D. , Gounari Fotini
Published: April 2021

Filensin and phakinin constitute the subunits of a heteropolymeric, lens-specific intermediate filament (IF) system known as the beaded-chain filaments (BFs). Since the rod of filensin is four heptads shorter than the rods of all other IF proteins, we decided to examine the specific contribution of this protein in filament assembly. For these purposes, we constructed chimeric proteins in which regions of filensin were exchanged with the equivalent ones of vimentin, a self-polymerizing IF protein. Our in vitro studies show that the filensin rod domain does not allow homopolymeric filament elongation. However, the filensin rod is necessary for co-polymerization of filensin with phakinin and seems to counteract the inherent tendency of the latter protein to homopolymerize into large, laterally associated filament bundles. Apart from the rod domain, the presence of an authentic or substituted tail domain in filensin is also essential for co-assembly with the naturally tail-less phakinin and formation of extended filaments in vitro. Finally, transfection experiments in CHO and MCF-7 cells show that the rod domain of filensin plays an important role in de novo filament formation and distribution. The same type of analysis further suggests that the end-domains of filensin interact with cell-specific, assembly-modulating factors.
MAP-1B/TAU functional redundancy during laminin-enhanced axonal growth
Authors: DiTella M. C. , Feiguin F. , Carri N. , Kosik K. S. , Cáceres A.
Published: April 2021

When cultured cerebellar macroneurons develop attached to a laminin-containing substrate or after the acute addition of laminin to the tissue culture medium, there is an acceleration in the rate and extent of axonal elongation. Furthermore, laminin is capable of inducing axonal formation and microtubule stabilization in neurons arrested at stage II of neuritic development by tau suppression (Caceres and Kosik, 1990; Caceres et al., 1991). Laminin-enhanced or induced axonal extension is paralleled by a selective and dramatic incorporation of phosphorylated MAP-1b into axonal microtubules. Axonal formation in neurons growing in the presence of laminin is prevented by treatment of the cultures with a mixture of MAP-1b and tau antisense oligonucleotides, but not by the single suppression of any one of these MAPs. However, suppression of MAP-1b, but not of tau, greatly reduces the increase in the rate and extent of axonal elongation induced by laminin. No such effects are elicited by MAP-1b antisense oligonucleotides in neurons growing in the absence of laminin, e.g. polylysine alone, where most of the MAP-1b present in the cells is dephosphorylated and not associated with the cytoskeleton. Taken collectively, these data suggest that, with regard to axonal elongation, MAP-1b and tau can be functionally substituted, and that extracellular matrix molecules, such as laminin, affect axonal extension by promoting the in vivo utilization of MAP-1b.
Cortical actin movements during the first cell cycle of the Caenorhabditis elegans embryo
Authors: Hird Steven
Published: April 2021

The first division of the Caenorhabditis elegans embryo is unequal, generating daughter cells with distinct fates. The differences between the cells are believed to result from the partitioning of cytoplasmic determinants during the first cell cycle. Actin microfilaments play a critical, but poorly defined, role in this event. In this paper, the actin cortex in live embryos is studied during cytoplasmic localisation by fluorescently labelling microfilaments in oocytes and then using in vivo fluorescence microscopy to observe their behaviour. This reveals that there is a concerted movement of cortical actin to the anterior of the embryo at the time cytoplasmic localisation takes place. Furthermore, it is demonstrated that endogenous foci of F-actin are asymmetrically distributed following this event; these structures have previously been seen in fixed cortices. A model for the participation of the actin cytoskeleton in cytoplasmic localisation is presented based on these results.
Autonomous differentiation in the mouse myogenic cell line, C2, involves a mutual positive control between insulin-like growth factor II and MyoD, operating as early as at the myoblast stage
Authors: Montarras Didier , Aurade Frédéric , Johnson Thomas , Ilan Joseph , Gros François , Pinset Christian
Published: April 2021

We have studied the contribution of the endogenous production of insulin-like growth factor II (IGFII) and of the muscle regulatory factor, MyoD, to the autonomy of differentiation in isolated skeletal myoblasts. Inhibition of MyoD and IGFII gene expression in myoblasts of the mouse myogenic cell line, C2, was achieved by transfection and selection of stably transfected cells (anti-MyoD and anti-IGFII cells) with vectors producing MyoD or IGFII antisense RNA. We observed that inhibiting either MyoD or IGFII has multiple and similar consequences. In addition to the inhibition of the target gene, expression of MyoD transcripts in anti-IGFII myoblasts and expression of IGFII in anti-MyoD myoblasts were also abolished, whereas accumulation of transcripts for the muscle regulatory factor, Myf5, was markedly increased in both cell types. However, despite this Myf5 up-regulation, both anti-IGFII and anti-MyoD myoblasts lost the ability to undergo autonomous differentiation (differentiation in the absence of added IGF), further indicating that Myf5 and MyoD are not strictly interchangeable. Additional evidence of a link between MyoD and IGFII was obtained: (1) forced expression of the MyoD cDNA stimulated IGFII gene expression, and (2) treatment of C2 myoblasts with fibroblast growth factor, not only diminished MyoD expression and compromised differentiation as previously shown by others, but also abolished IGFII expression. These experiments showing loss or gain of function argue in favor of a mutual positive control between IGFII and MyoD operating as early as the myoblast stage.
Tenascin mediates human glioma cell migration and modulates cell migration on fibronectin
Authors: Deryugina Elena I. , Bourdon Mario A.
Published: April 2021

The role of tenascin in mediating tumor cell migration was studied using two cell migration models. In migration/invasion Transwell assays U251.3 glioma cells rapidly migrated through the 8 µm pore size membranes onto tenascinand fibronectin-coated surfaces. In this assay the number of cells migrating onto tenascin was 52.2±9.6% greater than on fibronectin within 4 hours. To assess cell migration rates and cell morphology, U251.3 migration was examined in a two-dimension spheroid outgrowth assay. The radial distance migrated by U251.3 cells from tumor spheroids was found to be 53.8±4.9% greater on tenascin than on fibronectin. Cells migrating on tenascin display a very motile appearance, while cells migrating on fibronectin spread and maintain close intercellular contacts. Cell migration in the presence of integrin blocking antibodies demonstrated that migration on tenascin and fibronectin is mediated by distinct integrins, α2β1 and αvβ5/αvβ3, respectively. Since tenascin is coexpressed in malignant tumor matrices with fibronectin, we assessed the effects of tenascin on U251.3 cell migration mediated by fibronectin. Tenascin was found to provide a positive effect on fibronectin-mediated migration by altering cell morphology and enhancing cell motility. These effects of tenascin on fibronectin-mediated cell migration were inhibited by blocking β1 and α2β1integrins. The results suggest that tenascin may play a significant role in promoting tumor cell migration and invasiveness by modulating cell responses to normal matrix components.
Mechanism of endothelial cell shape change and cytoskeletal remodeling in response to fluid shear stress
Authors: Malek Adel M. , Izumo Seigo
Published: April 2021

Endothelium exposed to fluid shear stress (FSS) undergoes cell shape change, alignment and microfilament network remodeling in the direction of flow by an unknown mechanism. In this study we explore the role of tyrosine kinase (TK) activity, intracellular calcium ([Ca2+]i), mechanosensitive channels and cytoskeleton in the mechanism of cell shape change and actin stress fiber induction in bovine aortic endothelium (BAE). We report that FSS induces β-actin mRNA in a time- and magnitudedependent fashion. Treatment with quin2-AM to chelate intracellular calcium release and herbimycin A to inhibit TK activity abolished BAE shape change and actin stress fiber induction by FSS, while inhibition of protein kinase C with chelerythrine had no effect. Altering intermediate filament structure with acrylamide did not affect alignment or F-actin induction by FSS. Examining the role of the BAE cytoskeleton revealed a critical role for microtubules (MT). MT disruption with nocodazole blocked both FSS-induced morphological change and actin stress fiber induction. In contrast, MT hyperpolymerization with taxol attenuated the cell shape change but did not prevent actin stress fiber induction under flow. Mechanosensitive channels were found not to be involved in the FSS-induced shape change. Blocking the shear-activated current (IK.S) with barium and the stretch-activated cation channels (ISA) with gadolinium had no effect on the shear-induced changes in morphology and cytoskeleton. In summary, FSS has a profound effect on endothelial shape and F-actin network by a mechanism which depends on TK activity, intracellular calcium, and an intact microtubule network, but is independent of protein kinase C, intermediate filaments and shear- and stretch-activated mechanosensitive channels.
Direct observation and quantification of macrophage chemoattraction to the growth factor CSF-1
Authors: Webb Sarah E. , Pollard Jeffrey W. , Jones Gareth E.
Published: April 2021

The cloned mouse macrophage cell line, BAC1.25F, resembles primary macrophages in its dependence on colony stimulating factor-1 (CSF-1) for both viability and proliferation. Re-addition of CSF-1 to cytokine-deprived cells, which are rounded with diffusely organised F-actin, stimulates rapid cell spreading and cell polarisation. Using the Dunn chemotaxis chamber the movement of stimulated macrophages was monitored over a 2 hour period. Cells re stimulated with 1.32 nM human recombinant CSF-1 migrated at a mean rate of 7.71 µm per hour, but showed no directional preferences. In a linear concentration gradient of CSF-1, cytokine-deprived cells were again stim ulated to migrate and the mean rate of cell motility, at 6.88 µm per hour, was not significantly different from that measured in an isotropic environment of CSF-1. However, there was a strong preference for the cells to orientate so that their long axes aligned with the CSF-1 gradient and they migrated preferentially towards the source of CSF-1. Migrating cells contained abundant F-actin within the leading lamellae as judged by confocal imaging of fluor escent phalloidin, but the actin was not arranged into stress fibre-like structures. These data support the proposition that CSF-1 is both a chemokinetic and chemotactic agent for macrophages. Tumour necrosis factor (TNF-α) failed to stimulate cell migration and thus was neither chemokinetic nor a chemotactic agent. However, cells exposed to a dual concentration gradient of both TNF-α and CSF-1 did migrate successfully, although the chemotactic response to CSF-1 was abolished.
The relative roles of specific N- and C-terminal phosphorylation sites in the disassembly of intermediate filament in mitotic BHK-21 cells
Authors: Chou Ying-Hao , Opal Puneet , Quinlan Roy A. , Goldman Robert D.
Published: April 2021

Previously we identified p34cdc2 as one of two protein kinases mediating the hyperphosphorylation and disas sembly of vimentin in mitotic BHK-21 cells. In this paper, we identify the second kinase as a 37 kDa protein. This p37 protein kinase phosphorylates vimentin on two adjacent residues (thr-457 and ser-458) which are located in the C-terminal non-alpha-helical domain. Contrary to the p34cdc2 mediated N-terminal phosphorylation (at ser-55) which can disassemble vimentin intermediate filaments (IF) in vitro, p37 protein kinase phosphorylates vimentin-IF without obviously affecting its structure in vitro. We have further examined the in vivo role(s) of vimentin phosphorylation in the disassembly of the IF network in mitotic BHK cells by transient transfection assays. In untransfected BHK cells, the interphase vimentin IF networks are disassembled into non-filamentous aggregates when cells enter mitosis.

Transfection of cells with vimentin cDNA lacking the p34cdc2 phosphorylation site (ser55:ala) effectively prevents mitotic cells from disassembling their IF. In contrast, apparently normal disassembly takes place in cells trans fected with cDNA containing mutated p37 kinase phos phorylation sites (thr457:ala/ser458:ala). Transfection of cells with vimentin cDNAs lacking both the N- and C-terminal phosphorylation sites yields a phenotype indistin guishable from that obtained with the single N-terminal mutant. Taken together, our results demonstrate that the site-specific phosphorylation of the N-terminal domain, but not the C-terminal domain of vimentin plays an important role in determining the state of IF polymerization and supramolecular organization in mitotic cells.
sp42, the boar sperm tyrosine kinase, is a male germ cell-specific product with a highly conserved tissue expression extending to other mammalian species
Authors: Berruti Giovanna , Borgonovo Barbara
Published: April 2021

sp42, a tyrosine kinase of 42 kDa originally found in ejaculated boar spermatozoa, is so far the only tyrosine protein kinase to have been purified from mature male germ cells. We have developed and characterized rabbit polyclonal antibodies specifically directed against the boar sperm enzyme, which has been here purified to homogeneity. Anti-sp42 serum and sp42 affinity-purified antibodies work very well in western blot, immunoprecipitation and immunocytochemistry, and do not inhibit sp42 catalytic activity. Immunoblotting analyses reveal the presence of sp42 both in maturing boar epididymal (caput, corpus and cauda segment) spermatozoa and in testicular spermatogenic cells, thus establishing that the protein is effectively expressed in the germ cells and is not a sperm-associated protein secreted by the epididymal epithelium or male accessory glands. This finding is further strengthened by the fact that sp42 is not glycosylated, since different lectins fail to bind to sp42 and treatment of sp42 with different deglycosylation enzymes does not result in a reduction of the molecular mass of sp42. When different boar tissues are immunoscreened in western blot analysis, the results are all sp42-negative. The extension of the study to other mammalian species (human, mouse and rat) demonstrates that proteins immunologically related to boar sp42, which share the same molecular mass and tyrosine kinase activity, are both expressed in spermatogenic cells and maintained in mature sperm cells. Intriguingly, when a wide spectrum of somatic mouse and rat tissues is probed with sp42-antiserum, no tissue presents anti-sp42 immunoreactivity. Immunocytochemistry shows that in boar spermatozoa sp42 is confined to the tail mid-piece, while by immunohistochemistry carried out on sections of adult rat testis the appearance time of the kinase appears to be consistent with a post-meiotic synthesis in haploid spermatids. Altogether, these results demonstrate that boar sp42 is a new male germ cell-specific gene product, with highly conserved tissue expression extended to other mammalian species, and suggest a possible role played by the cytoplasmic tyrosine kinase in the cell signalling network specific to haploid male germ cells.
An ultradian clock controls locomotor behaviour and cell division in isolated cells of Paramecium tetraurelia
Authors: Kippert Fred
Published: April 2021

An ultradian clock operates in fast growing cells of the large ciliate, Paramecium tetraurelia. The period of around 70 minutes is well temperature-compensated over the temperature range tested, i.e. between 18°C and 33°C. The Q10 between 18°C and 27°C is 1.08; above 27°C there is a slight overcompensation. The investigation of individual cells has revealed that two different cellular functions are under temporal control by this ultradian clock. First, locomotor behaviour, which is an alternation between a phase of fast swimming with only infrequent turning, and a phase of slow swimming with frequent spontaneous changes of direction. In addition, the ultradian clock is involved in the timing of cell division. Generation times are not randomly distributed, but occur in well separated clusters. At all of the six temperatures tested, the clusters are separated by around 70 minutes which corresponds well to the period of the locomotor behaviour rhythm at the respective temperatures. Whereas the interdivision times were gradually lengthened both above and below the optimum growth temperature, the underlying periodicity remained unaffected. Also cells of different clonal age had identical periods, suggesting that neither the differences in DNA content, not other changes associated with ageing in Paramecium have an effect on the clock. A constant phase relationship was observed between the rhythm in locomotor behaviour and the time window for cell division; this strongly suggests that the same ultradian clock exerts temporal control over both processes.
Antisense RNA inactivation of gene expression of a cellcell adhesion protein (gp64) in the cellular slime mold Polysphondylium pallidum
Authors: Funamoto Satoru , Ochiai Hiroshi
Published: April 2021

The gp64 protein of Polysphondylium pallidum has been shown to mediate EDTA-stable cellcell adhesion. To explore the functional role of gp64, we made an antisense RNA expression construct designed to prevent the gene expression of gp64; the construct was introduced into P. pallidum cells and the transformants were characterised. The antisense RNA-expressing clone L3mc2 which had just been harvested at the growth phase tended to reform in aggregates smaller in size than did the parental cells in either the presence or absence of 10 mM EDTA. In contrast, 6.5-hour starved L3mc2 cells remained considerably dissociated from each other after 5 minutes gyrating, although aggregation gradually increased by 50% during a further 55 minutes gyrating, in the presence of 10 mM EDTA. Correspondingly, L3mc2 lacked specifically the cellcell adhesion protein, gp64. We therefore conclude that the gp64 protein is involved in forming the EDTA-resistant cellcell contact. In spite of the absence of gp64, L3mc2 exhibited normal developmental processes, a fact which demonstrates that another cellcell adhesion system exists in the development of Polysphondylium. This is the first report in which an antisense RNA technique was successfully applied to Polysphondylium.
Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage
Authors: Michel M. , Torok N. , Godbout M.J. , Lussier M. , Gaudreau P. , Royal A. , Germain L.
Published: April 2021

This study was undertaken to evaluate keratin 19 (K19) as a biochemical marker for skin stem cells in order to address some long standing questions concerning these cells in the field of cutaneous biology. We first used the well-established mouse model enabling us to identify skin stem cells as [3H]thymidine-label-retaining cells. A site directed antibody was raised against a synthetic peptide of K19. It reacted specifically with a 40 kDa protein (K19) on immunoblotting. It labelled the bulge area of the outer root sheath on mouse skin by immunohistochemistry. Double-labelling revealed that K19-positive-cells were also [3H]thymidine-label-retaining cells, suggesting that K19 is a marker for skin stem cells of hair follicles. K19-expression was then used to investigate the variation in mouse and human skin stem cells as a function of body site, donor age and culture time. K19 was expressed in the hair follicle and absent from the interfollicular epidermis at hairy sites (except for some K18 coexpressing Merkel cells). In contrast, at glabrous sites, K19-positive-cells were in deep epidermal rete ridges. K19 expressing cells also contained high levels of alpha 3 beta 1 integrin. The proportion of K19-positive-cells was greater in newborn than older foreskins. This correlated with keratinocyte culture lifespan variation with donor age. Moreover, it could explain clinical observations that children heal faster than adults. In conclusion, K19 expression in skin provides an additional tool to allow further characterization of skin stem cells under normal and pathological conditions in situ and in vitro.
Presence and expression of G2 cyclins in the coelenterate hydra
Authors: Scheurlen Imke , Hoffmeister Sabine A. H. , Schaller H. Chica
Published: April 2021

In hydra all cellcycle control occurs in the G2/M transition. Cyclins acting at this restriction point in the cell cycle belong to the cyclin A and B families. In agreement with this we isolated cDNAs coding for a cyclin A and a cyclin B from the multiheaded mutant of Chlorohydra viridissima and a cyclin B from Hydra vulgaris. The two B-type cyclins from hydra show 85.6% identity at the amino acid level, and 84.8% at the nucleotide level. The relatedness is less extensive than that found for mammals, e.g. human and mouse, and is evidence that the two hydra species diverged early in evolution. From each hydra species only one B-type cyclin was found, showing equal relatedness to the B1 and B2 subtypes of cyclins, hinting at a role as common ancestor before the split into B1 and B2 cyclins occurred. All three hydra cyclins contain regulation signals typical for G2/M cyclins, such as a ubiquitin destruction box at the amino terminus, needed for rapid degradation of the protein, and translation and polyadenylation elements in the 3’ untranslated region to regulate RNA storage and RNA degradation.

In hydra cellcycle times vary depending on feeding regime and growth conditions. Cyclin B RNA expression was found to precede the daily mitotic rhythm induced by feeding. During head regeneration cyclin B expression showed the expected drop early during regeneration and an increase later. At the cellular level strongest expression of cyclin B RNA and protein was detected in interstitial cells which possess with one day the shortest cellcycle time in hydra. Epithelial cells with a threeday cellcycle rhythm showed variable, and differentiated cells no cyclin B expression. Regions of hydra containing high numbers of proliferating cells, such as developing buds exhibited elevated levels of cyclin B expression.
Plakoglobin domains that define its association with the desmosomal cadherins and the classical cadherins: Identification of unique and shared domains
Authors: Wahl James K. , Sacco Paula A. , Sadler Tammy M. McGranahan- , Sauppé Laura M. , Wheelock Margaret J. , Johnson Keith R.
Published: April 2021

Two cell-cell junctions, the adherens junction and the desmosome, are prominent in epithelial cells. These junctions are composed of transmembrane cadherins which interact with cytoplasmic proteins that serve to link the cadherin to the cytoskeleton. One component of both adherens junctions and desmosomes is plakoglobin. In the adherens junction plakoglobin interacts with both the classical cadherin and with αcatenin. Alphacatenin in turn interacts with microfilaments. The role plakoglobin plays in the desmosome is not well understood. Plakoglobin interacts with the desmosomal cadherins, but how and if this mediates interactions with the intermediate filament cytoskeleton is not known. Here we compare the domains of plakoglobin that allow it to associate with the desmosomal cadherins with those involved in interactions with the classical cadherins. We show that three sites on plakoglobin are involved in associations with the desmosomal cadherins. A domain near the N terminus is unique to the desmosomal cadherins and overlaps with the site that interacts with αcatenin, suggesting that there may be competition between αcatenin and the desmosomal cadherins for interactions with plakoglobin. In addition, a central domain is shared with regions used by plakoglobin to associate with the classical cadherins. Finally, a domain near the C terminus is shown to strongly modulate the interactions with the desmosomal cadherins. This latter domain also contributes to the association of plakoglobin with the classical cadherins.
Organelle motility and metabolism in axons vs dendrites of cultured hippocampal neurons
Authors: Overly Caroline C. , Rieff Heather I. , Hollenbeck Peter J.
Published: April 2021

Regional regulation of organelle transport seems likely to play an important role in establishing and maintaining distinct axonal and dendritic domains in neurons, and in managing differences in local metabolic demands. In addition, known differences in microtubule polarity and organization between axons and dendrites along with the directional selectivity of microtubulebased motor proteins suggest that patterns of organelle transport may differ in these two process types. To test this hypothesis, we compared the patterns of movement of different organelle classes in axons and different dendritic regions of cultured embryonic rat hippocampal neurons. We first examined the net direction of organelle transport in axons, proximal dendrites and distal dendrites by videoenhanced phasecontrast microscopy. We found significant regional variation in the net transport of large phasedense vesicular organelles: they exhibited net retrograde transport in axons and distal dendrites, whereas they moved equally in both directions in proximal dendrites. No significant regional variation was found in the net transport of mitochondria or macropinosomes. Analysis of individual organelle motility revealed three additional differences in organelle transport between the two process types. First, in addition to the difference in net transport direction, the large phasedense organelles exhibited more persistent changes in direction in proximal dendrites where microtubule polarity is mixed than in axons where microtubule polarity is uniform. Second, while the net direction of mitochondrial transport was similar in both processes, twice as many mitochondria were motile in axons than in dendrites. Third, the mean excursion length of moving mitochondria was significantly longer in axons than in dendrites. To determine whether there were regional differences in metabolic activity that might account for these motility differences, we labeled mitochondria with the vital dye, JC-1, which reveals differences in mitochondrial transmembrane potential. Staining of neurons with this dye revealed a greater proportion of highly charged, more metabolically active, mitochondria in dendrites than in axons. Together, our data reveal differences in organelle motility and metabolic properties in axons and dendrites of cultured hippocampal neurons.
Tubulin folding is altered by mutations in a putative GTP binding motif
Authors: Zabala Juan C. , Fontalba Ana , Avila Jesus
Published: April 2021

Tubulins contain a glycine-rich loop, that has been implicated in microtubule dynamics by means of an intramolecular interaction with the carboxy-terminal region. As a further extension of the analysis of the role of the carboxy-terminal region in tubulin folding we have mutated the glycine-rich loop of tubulin subunits. An α-tubulin point mutant with a T150rG substitution (the corresponding residue present in β-tubulin) was able to incorporate into dimers and microtubules. On the other hand, four β-tubulin point mutants, including the G148rT substitution, did not incorporate into dimers, did not release monomers, but were able to form C900 and C300 complexes (interme-diates in the process of tubulin folding). Three other mutants within this region (which approximately encom-passes residues 137-152) were incapable of forming dimers and C300 complexes but gave rise to the formation of C900 complexes. These results suggest that tubulin goes through two sequential folding states during the folding process, first in association with TCP1-complexes (C900) prior to the transfer to C300 complexes. It is this second step that implies binding/hydrolysis of GTP, reinforcing our previous proposed model for tubulin folding and assembly.
Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum
Authors: Temesvari Lesly A. , Rodriguez-Paris Juan M. , Bush John M. , Zhang Linyi , Cardelli James A.
Published: April 2021

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H+-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989)J. Biol. Chem. 264, 3454-3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, α-mannosidase. CMA treatment com-promised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. dis-coideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H+-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H+-ATPase.
Blackjack, a novel protein associated with microtubules in embryonic neurons
Authors: Zachow Karen R. , Bentley David
Published: April 2021

Microtubule-associated proteins can influence the organization, stability and dynamics of microtubules. We characterize a novel protein that associates with microtubules as assessed by immunofluorescence, immunoelectron microscopy, and co-sedimentation. The protein is expressed heavily in embryonic neurons and, to a lesser extent, in epithelial and mesodermal cells. The cDNA sequence predicts a protein of 1,547 amino acids and approximately 170 kDa. Immunoblot of embryo lysate demonstrates bands of approximately 240 and 260 kDa. The predicted amino acid sequence contains 77 potential serine/threonine phosphorylation sites. A distinctive feature is a predicted α-helical central domain comprising 21 identical repeats of an 11 amino acid sequence (PLEELRKDAAE). The protein is thermostable and has two major charge-domains: the amino-terminal 80% has an estimated pI of 4.0 and the carboxy-terminal 20%, a pI of 12.2. The protein shares several general biochemical and molecular features of MAPs, but its sequence is not similar to that of any described MAP.
Interferon-independent activation of (2′-5′) oligoadenylate synthetase in Friend erythroleukemia cell variants exposed to HMBA
Authors: Salzberg Samuel , Heller Adi , Zou Jian-Ping , Collart Frank R. , Huberman Eliezer
Published: April 2021

To provide evidence for the implication of interferon (IFN)-induced proteins in the regulation of cell growth during differentiation, the activation of (2′-5′) oligoadenylate synthetase (2-5A synthetase) as well as of PKR, two IFN-induced proteins, during differentiation of Friend erythroleukemia cells, was studied. Two cell variants were used. The first (FL) was completely susceptible to hexamethylene bis-acetamide (HMBA)-treatment and responded in both growth-retardation and hemoglobin synthesis. The second (R1) failed to synthesize hemoglobin in response to HMBA although cell growth was still inhibited. In both cell variants, 2-5A synthetase enzyme activity was induced in a similar fashion, reaching a peak at 26 hours after treatment with HMBA. However, the down regulation of activity thereafter was not identical in both cases. In R1 cells, the reduction was much slower compared to FL cells. A similar pattern was observed with the appearance of the 43 kDa isoform of 2-5A synthetase in immunoblots. An analysis of 2-5A synthetase gene expression revealed the presence of 1.7 kb transcripts which peaked at 16 hours after HMBA-treatment in both cell variants. Again, the down-regulation in expression was slower in R1 than in FL cells. Addition of anti-murine α/β-IFN antibodies did not reduce the level of either 2-5A synthetase expression or enzyme activity in either cell variant. Interestingly, the presence of antibodies also did not affect the pattern of pRb phosphorylation in the cell variants exposed to HMBA. In both cell variants, an increase in the amount of the phosphorylated form (ppRb) was observed in immunoblots after 4 hours. This form was gradually trans-formed to the underphosphorylated molecule (pRb) with time in culture, even in the presence of antibodies. This further substantiates the notion that IFN-induced regulation of pRb phosphorylation is mediated by IFN-induced proteins.

The basal level of either expression or ezymatic activity of PKR detected in untreated FL or R1 cells, was relatively high. Treatment with HMBA did not result in further induction of PKR in either cell variant.