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See detailA microtubule-independent role of p150glued in secretory cargo concentration at endoplasmic reticulum exit sites
Verissimo, Fatima; Halavatyi, Aliaksandr UL; Pepperkok, Rainer et al

in Journal of Cell Science (2015), 128(22), 4160-4170

Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by ... [more ▼]

Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by membrane association and GTP loading of SAR1, followed by the recruitment of the SEC23-SEC24 and SEC13-SEC31 subcomplexes. Both of these two subcomplexes stimulate GTP hydrolysis and coat disassembly. This inherent disassembly capacity of COPII complexes needs to be regulated to allow sufficient time for cargo sorting and transport carrier formation. By performing fluorescence recovery after photobleaching (FRAP) and mathematical modeling, we show that p150glued (also known as DCTN1), a component of the dynactin complex, stabilizes the COPII pre-budding complex on ER membranes in a microtubule-independent manner. Concentration of the secretory marker ts-O45-G at ERES is reduced in the presence of a C-terminal p150glued fragment that prevents binding of endogenous p150glued to SEC23. A similar cargo reduction is observed upon p150glued knockdown. Taken together, our data suggest that cargo concentration at ERES is regulated by p150glued to coordinate protein sorting and transport carrier formation with the subsequent long-range transport towards the Golgi complex along microtubules. © 2015. Published by The Company of Biologists Ltd. [less ▲]

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See detailCross-regulation of cytokine signalling: Pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation
Radtke, S.; Wüller, S.; Yang, X.-P. et al

in Journal of Cell Science (2010), 123(6), 947-959

The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on ... [more ▼]

The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1b, TNFa or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response. [less ▲]

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See detailThe stimulation of dendrite growth by Sema3A requires integrin engagement and focal adhesion kinase.
Schlomann, Uwe; Schwamborn, Jens Christian UL; Muller, Myriam et al

in Journal of Cell Science (2009), 122(Pt 12), 2034-42

The rate and direction of axon and dendrite growth depend on multiple guidance signals and growth factors. Semaphorin 3A (Sema3A) acts as a repellent for axons and attractant for dendrites. Here, we show ... [more ▼]

The rate and direction of axon and dendrite growth depend on multiple guidance signals and growth factors. Semaphorin 3A (Sema3A) acts as a repellent for axons and attractant for dendrites. Here, we show that the requirement for integrin engagement distinguishes the response of axons and dendrites to Sema3A in hippocampal neurons. Sema3A promotes the extension of hippocampal dendrites by a pathway that requires focal adhesion kinase (FAK). The stimulation of dendrite growth and FAK phosphorylation by Sema3A depend on integrin engagement. Unlike their function as a target of Sema3A during the collapse of axonal growth cones, integrins facilitate the stimulation of dendrite extension. Conditional inactivation of the genes encoding beta1 integrin or FAK blocks the growth-promoting effect of Sema3A but not the collapse of axonal growth cones. Our results demonstrate that different pathways mediate the stimulation of dendrite growth and the collapse of axonal growth cones by Sema3A. [less ▲]

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See detailNucleocytoplasmic shuttling of persistently activated STAT3.
Herrmann, Andreas; Vogt, Michael; Monnigmann, Martin et al

in Journal of Cell Science (2007), 120(Pt 18), 3249-61

Persistent activation of the transcription factor STAT3 has been detected in many types of cancer and plays an important role in tumor progression, immune evasion and metastasis. To analyze persistent ... [more ▼]

Persistent activation of the transcription factor STAT3 has been detected in many types of cancer and plays an important role in tumor progression, immune evasion and metastasis. To analyze persistent STAT3 activation we coexpressed STAT3 with v-Src. We found that tyrosine phosphorylation of STAT3 by v-Src is independent of Janus kinases (Jaks), the canonical activators of STATs. The STAT3-induced feedback inhibitor, suppressor of cytokine signaling 3 (SOCS3), did not interfere with STAT3 activation by v-Src. However, the protein inhibitor of activated STAT3 (PIAS3) suppressed gene induction by persistently activated STAT3. We measured nucleocytoplasmic shuttling of STAT3 in single cells by bleaching the YFP moiety of double-labelled STAT3-CFP-YFP in the cytoplasm. Analysis of the subcellular distribution of CFP and YFP fluorescence over time by mathematical modeling and computational parameter estimation revealed that activated STAT3 shuttles more rapidly than non-activated STAT3. Inhibition of exportin-1-mediated nuclear export slowed down nucleocytoplasmic shuttling of v-Src-activated STAT3 resulting in reduced tyrosine phosphorylation, decreased induction of STAT3 target genes and increased apoptosis. We propose passage of persistently activated STAT3 through the nuclear pore complex as a new target for intervention in cancer. [less ▲]

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See detailPhosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells
Janji, B.; Giganti, A.; De Corte, V. et al

in Journal of Cell Science (2006), 119(9), 1947-1960

L-plastin, a malignant transformation-associated protein, is a member of a large family of actin filament cross-linkers. Here, we analysed how phosphorylation of L-plastin on Ser5 of the headpiece domain ... [more ▼]

L-plastin, a malignant transformation-associated protein, is a member of a large family of actin filament cross-linkers. Here, we analysed how phosphorylation of L-plastin on Ser5 of the headpiece domain regulates its intracellular distribution and its interaction with F-actin in transfected cells and in in vitro assays. Phosphorylated wild-type L-plastin localised to the actin cytoskeleton in transfected Vero cells. Ser5Ala substitution reduced the capacity of L-plastin to localise with peripheral actin-rich membrane protrusions. Conversely, a Ser5Glu variant mimicking a constitutively phosphorylated state, accumulated in actin-rich regions and promoted the formation of F-actin microspikes in two cell lines. Similar to phosphorylated wild-type L-plastin, this variant remained associated with cellular F-actin in detergent-treated cells, whereas the Ser5Ala variant was almost completely extracted. When compared with non-phosphorylated protein, phosphorylated L-plastin and the Ser5Glu variant bound F-actin more efficiently in an in vitro assay. Importantly, expression of L-plastin elicited collagen invasion in HEK293T cells, in a manner dependent on Ser5 phosphorylation. Based on our findings, we propose that conversely to other calponin homology (CH)-domain family members, phosphorylation of L-plastin switches the protein from a low-activity to a high-activity state. Phosphorylated L-plastin might act as an integrator of signals controlling the assembly of the actin cytoskeleton and cell motility in a 3D-space. [less ▲]

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See detailActin-filament cross-linking protein T-plastin increases Arp2/3-mediated actin-based movement
Giganti, A.; Plastino, J.; Janji, B. et al

in Journal of Cell Science (2005), 118(6), 1255-1265

Increasing evidence suggests that actin cross-linking or bundling proteins might not only structure the cortical actin cytoskeleton but also control actin dynamics. Here, we analyse the effects of T ... [more ▼]

Increasing evidence suggests that actin cross-linking or bundling proteins might not only structure the cortical actin cytoskeleton but also control actin dynamics. Here, we analyse the effects of T-plastin/T-fimbrin, a representative member of an important actin-filament cross-linking protein by combining a quantitative biomimetic motility assay with biochemical and cell-based approaches. Beads coated with the VCA domain of the Wiskott/Aldrich-syndrome protein (WASP) recruit the actin-nucleating Arp2/3 complex, polymerize actin at their surface and undergo movement when placed in cell-free extracts. T-Plastin increased the velocity of VCA beads 1.5 times, stabilized actin comets and concomitantly displaced cofilin, an actin-depolymerizing protein. T-Plastin also decreased the F-actin disassembly rate and inhibited cofilin-mediated depolymerization of actin filaments in vitro. Importantly, a bundling-incompetent variant comprising the first actin-binding domain (ABD1) had similar effects. In cells, this domain induced the formation of long actin cables to which other actin-regulating proteins were recruited. Altogether, these results favor a mechanism in which binding of ABD1 controls actin turnover independently of cross-link formation. In vivo, this activity might contribute to the assembly and maintenance of the actin cytoskeleton of plasma-membrane protrusions. [less ▲]

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See detailSTAT3 is enriched in nuclear bodies.
Herrmann, Andreas; Sommer, Ulrike; Pranada, Albert L. et al

in Journal of Cell Science (2004), 117(Pt 2), 339-49

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is involved in a variety of biological functions. It is essential for the signal transduction of interleukin-6 (IL ... [more ▼]

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is involved in a variety of biological functions. It is essential for the signal transduction of interleukin-6 (IL-6) and related cytokines. In response to IL-6 stimulation STAT3 becomes phosphorylated and translocates into the nucleus where it binds to enhancer sequences of target genes. We found that activated STAT3 is enriched in dot-like structures within the nucleus, which we termed STAT3 nuclear bodies. To examine the dynamics of STAT3 nuclear body formation, a fusion protein of STAT3 and yellow fluorescent protein (YFP) was constructed. Studies in living cells have shown that the appearance of STAT3 nuclear bodies is transient, correlating with the timecourse of tyrosine-phosphorylation of STAT3. Furthermore, we show by fluorescence recovery after photobleaching (FRAP) analysis that STAT3 within nuclear bodies consists of a highly mobile and an immobile fraction. Colocalization studies provided evidence that these bodies are accompanied with CREB binding protein (CBP) and acetylated histone H4, which are markers for transcriptionally active chromatin. Moreover, STAT3 nuclear bodies in HepG2 cells are not colocalized with promyelocytic leukemia oncoprotein (PML)-containing bodies; neither is a sumoylation of activated STAT3 detectable. Taken together, our data suggest that STAT3 nuclear bodies are either directly involved in active gene transcription or they serve as reservoirs of activated STAT3. [less ▲]

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See detailStructural and functional similarities between the human cytoskeletal protein zyxin and the ActA protein of Listeria monocytogenes.
Golsteyn, R. M.; Beckerle, M. C.; Koay, T. et al

in Journal of cell science (1997), 110 ( Pt 16)

The intracellular bacterial parasite Listeria monocytogenes produces ActA protein at its surface to facilitate the localized assembly of actin-filled comets that are required for movement. The ... [more ▼]

The intracellular bacterial parasite Listeria monocytogenes produces ActA protein at its surface to facilitate the localized assembly of actin-filled comets that are required for movement. The organization of actin in Listeria comets shows striking similarity to the organization of actin at the plasma membrane of mammalian cells. Therefore we examined the possibility that an ActA-like protein is present in mammalian cells. By using antibodies directed against ActA, we identified zyxin as an ActA related protein in a number of cell types. We compared the functions of ActA and zyxin by transient expression of variants tagged with an inner plasma membrane localization sequence (a CAAX box). Targeting of the proline rich domain of zyxin to the plasma membrane disrupts the actin cytoskeleton and cell shape in a manner similar to that which occurs with membrane-targeted ActA sequences. A chimeric protein composed of the N-terminal domain of ActA fused to the N-terminal and central domains of zyxin induced a full ActA response in cells. Furthermore, zyxin and ActA exhibit common protein partners in vitro. On the basis of the shared properties of zyxin and ActA, we propose that zyxin enhances actin organizing activity in mammalian cells. [less ▲]

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See detailVillin-induced growth of microvilli is reversibly inhibited by cytochalasin D.
Friederich, Evelyne UL; Kreis, T. E.; Louvard, D.

in Journal of cell science (1993), 105 ( Pt 3)

Villin is an actin-binding protein that is associated with the cytoskeleton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca(2+)-dependent manner. In the ... [more ▼]

Villin is an actin-binding protein that is associated with the cytoskeleton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca(2+)-dependent manner. In the absence of Ca2+, villin organizes microfilaments into bundles. Transfection of a villin-specific cDNA into cultured cells that do not produce this protein results in the growth of long surface microvilli and the reorganization of the underlying actin cytoskeleton. Here we studied the effects of low concentrations of cytochalasin D on the induction of these plasma membrane-actin cytoskeleton specializations. Transfected cells were treated with concentrations of cytochalasin D that prevent the association of actin monomers with the fast-growing end of microfilaments in vitro. In villin-positive cells, cytochalasin D inhibited the growth of microvilli and promoted the formation of rodlet-like actin structures, which were randomly distributed throughout the cytoplasm. The formation of these structures was dependent on large amounts of villin and on the integrity of an actin-binding site located at the carboxy terminus of villin, which is required for microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imaging revealed that when cytochalasin D was removed, rapid disassembly of actin rodlets occurred after a lag phase. The present data stress the important role of the plasma membrane in the organization of the actin cytoskeleton and suggest that the extension of the microvillar plasma membrane is dependent on the elongation of microfilaments at their fast-growing end. Inhibition of microfilament elongation near the plasma membrane by cytochalasin D may result in the 'random' nucleation of actin filaments throughout the cytoplasm. On the basis of the present data, we propose that villin is involved in the assembly of the microvillar actin bundle by a mechanism that does not prevent monomer association with the preferred end of microfilaments. For instance, villin may stabilize actin filaments by lateral interactions. The functional importance of the carboxy-terminal F-actin binding site in such a mechanism is stressed by the fact that it is required for the formation of F-actin rodlets in cytochalasin D-treated cells. Finally, our data further emphasize the observations that the effects of cytochalasin D in living cells can be modulated by actin-binding proteins. [less ▲]

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