A microtubule-independent role of p150glued in secretory cargo concentration at endoplasmic reticulum exit sites

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 ▲]

The stimulation of dendrite growth by Sema3A requires integrin engagement and focal adhesion kinase.

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 ▲]

Phosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells

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 ▲]

STAT3 is enriched in nuclear bodies.

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 ▲]

Villin-induced growth of microvilli is reversibly inhibited by cytochalasin 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 ▲]