24 research outputs found
Formation of filopodia-like bundles in vitro from a dendritic network
We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)–activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)–coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin
Phosphorylation controls autoinhibition of cytoplasmic linker protein-170
Author Posting. © American Society for Cell Biology, 2010. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 21 (2010): 2661-2673, doi:10.1091/mbc.E09-12-1036.Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150Glued (J. Cell Biol. 2004: 166, 1003–1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an "open" conformation and a higher binding affinity for growing MT ends and p150Glued as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the "folded back" conformation shows decreased MT association and does not interact with p150Glued. We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.This work was supported by National
Institutes of Health grant GM-25062 (to G.G.B.); Netherlands Organization for
Scientific Research grants (to A. A. and N. G.); a Cancer Genomics Centre
grant (to J.v.H.); and Presidential Program of Russian Academy of Sciences
and RFBP grant 05-04-4915 (to E.S.N.)
The SOLAS air-sea gas exchange experiment (SAGE) 2004
Author Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Deep Sea Research Part II: Topical Studies in Oceanography 58 (2011): 753-763, doi:10.1016/j.dsr2.2010.10.015.The SOLAS air-sea gas exchange experiment (SAGE) was a multiple-objective study investigating
gas-transfer processes and the influence of iron fertilisation on biologically driven gas exchange in
high-nitrate low-silicic acid low-chlorophyll (HNLSiLC) Sub-Antarctic waters characteristic of the
expansive Subpolar Zone of the southern oceans. This paper provides a general introduction and
summary of the main experimental findings. The release site was selected from a pre-voyage desktop
study of environmental parameters to be in the south-west Bounty Trough (46.5°S 172.5°E) to the
south-east of New Zealand and the experiment conducted between mid-March and mid-April 2004. In
common with other mesoscale iron addition experiments (FeAX’s), SAGE was designed as a
Lagrangian study quantifying key biological and physical drivers influencing the air-sea gas exchange
processes of CO2, DMS and other biogenic gases associated with an iron-induced phytoplankton
bloom. A dual tracer SF6/3He release enabled quantification of both the lateral evolution of a labelled
volume (patch) of ocean and the air-sea tracer exchange at the 10’s of km’s scale, in conjunction with
the iron fertilisation. Estimates from the dual-tracer experiment found a quadratic dependency of the
gas exchange coefficient on windspeed that is widely applicable and describes air-sea gas exchange in strong wind regimes. Within the patch, local and micrometeorological gas exchange process studies (100 m scale) and physical variables such as near-surface turbulence, temperature microstructure at the interface, wave properties, and wind speed were quantified to further assist the development of gas exchange models for high-wind environments.
There was a significant increase in the photosynthetic competence (Fv/Fm) of resident phytoplankton
within the first day following iron addition, but in contrast to other FeAX’s, rates of net primary
production and column-integrated chlorophyll a concentrations had only doubled relative to the
unfertilised surrounding waters by the end of the experiment. After 15 days and four iron additions
totalling 1.1 tonne Fe2+, this was a very modest response compared to the other mesoscale iron
enrichment experiments. An investigation of the factors limiting bloom development considered co-
limitation by light and other nutrients, the phytoplankton seed-stock and grazing regulation. Whilst
incident light levels and the initial Si:N ratio were the lowest recorded in all FeAX’s to date, there was
only a small seed-stock of diatoms (less than 1% of biomass) and the main response to iron addition
was by the picophytoplankton. A high rate of dilution of the fertilised patch relative to phytoplankton
growth rate, the greater than expected depth of the surface mixed layer and microzooplankton grazing
were all considered as factors that prevented significant biomass accumulation. In line with the limited
response, the enhanced biological draw-down of pCO2 was small and masked by a general increase in pCO2 due to mixing with higher pCO2 waters. The DMS precursor DMSP was kept in check through grazing activity and in contrast to most FeAX’s dissolved dimethylsulfide (DMS) concentration declined through the experiment. SAGE is an important low-end member in the range of responses to iron addition in FeAX’s. In the context of iron fertilisation as a geoengineering tool for atmospheric CO2 removal, SAGE has clearly demonstrated that a significant proportion of the low iron ocean may not produce a phytoplankton bloom in response to iron addition.SAGE was jointly funded through
the New Zealand Foundation for Research, Science and Technology (FRST) programs
(C01X0204) "Drivers and Mitigation of Global Change" and (C01X0223) "Ocean
Ecosystems: Their Contribution to NZ Marine Productivity." Funding was also provided for
specific collaborations by the US National Science Foundation from grants OCE-0326814
(Ward), OCE-0327779 (Ho), and OCE 0327188 OCE-0326814 (Minnett) and the UK Natural
Environment Research Council NER/B/S/2003/00282 (Archer). The New Zealand
International Science and Technology (ISAT) linkages fund provided additional funding
(Archer and Ziolkowski), and the many collaborator institutions also provided valuable
support
Phosphorylation controls autoinhibition of cytoplasmic linker protein-170
Author Posting. © American Society for Cell Biology, 2010. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 21 (2010): 2661-2673, doi:10.1091/mbc.E09-12-1036.Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150Glued (J. Cell Biol. 2004: 166, 1003–1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an "open" conformation and a higher binding affinity for growing MT ends and p150Glued as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the "folded back" conformation shows decreased MT association and does not interact with p150Glued. We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.This work was supported by National
Institutes of Health grant GM-25062 (to G.G.B.); Netherlands Organization for
Scientific Research grants (to A. A. and N. G.); a Cancer Genomics Centre
grant (to J.v.H.); and Presidential Program of Russian Academy of Sciences
and RFBP grant 05-04-4915 (to E.S.N.)
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