Visualizing and Quantifying In Vivo Cortical Cytoskeleton Structure and Dynamics

Background: First developed in the 1990’s at the National Synchrotron Light Source, xray synchrotron footprinting is an ideal technique for the analysis of solution-state structure and dynamics of macromolecules. Hydroxyl radicals generated in aqueous samples by intense x-ray beams serve as fine probes of solvent accessibility, rapidly and irreversibly reacting with solvent exposed residues to provide a “snapshot” of the sample state at the time of exposure. Over the last few decades, improvements in instrumentation to expand the technology have continuously pushed the boundaries of biological systems that can be studied using the technique. Conclusion: Dedicated synchrotron beamlines provide important resources for examining fundamental biological mechanisms of folding, ligand binding, catalysis, transcription, translation, and macromolecular assembly. The legacy of synchrotron footprinting at NSLS has led to significant improvement in our understanding of many biological systems, from identifying key structural components in enzymes and transporters to in vivo studies of ribosome assembly. This work continues at the XFP (17-BM) beamline at NSLS-II and facilities at ALS, which are currently accepting proposals for use.

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Structure and Dynamics of the Glycolipid Components of Membrane Receptors:2H NMR Provides a Route to In Vivo Observation

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1989.tb12489.x ◽

1989 ◽

Vol 568 (1 Calcium, Memb) ◽

pp. 44-51

Author(s):

IAN C. P. SMITH ◽

MICHÈLE AUGER ◽

HAROLD C. JARRELL

Keyword(s):

Membrane Receptors ◽

2H Nmr ◽

Structure And Dynamics

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Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment

The Journal of Cell Biology ◽

10.1083/jcb.201005140 ◽

2010 ◽

Vol 191 (4) ◽

pp. 771-781 ◽

Cited By ~ 76

Author(s):

Alexander Ludwig ◽

Grant P. Otto ◽

Kirsi Riento ◽

Emily Hams ◽

Padraic G. Fallon ◽

...

Keyword(s):

Plasma Membrane ◽

Ex Vivo ◽

Neutrophil Migration ◽

Neutrophil Recruitment ◽

Membrane Microdomains ◽

Neutrophil Chemotaxis ◽

Myosin Iia ◽

Plasma Membrane Microdomains ◽

Cortical Cytoskeleton

We studied the function of plasma membrane microdomains defined by the proteins flotillin 1 and flotillin 2 in uropod formation and neutrophil chemotaxis. Flotillins become concentrated in the uropod of neutrophils after exposure to chemoattractants such as N-formyl-Met-Leu-Phe (fMLP). Here, we show that mice lacking flotillin 1 do not have flotillin microdomains, and that recruitment of neutrophils toward fMLP in vivo is reduced in these mice. Ex vivo, migration of neutrophils through a resistive matrix is reduced in the absence of flotillin microdomains, but the machinery required for sensing chemoattractant functions normally. Flotillin microdomains specifically associate with myosin IIa, and spectrins. Both uropod formation and myosin IIa activity are compromised in flotillin 1 knockout neutrophils. We conclude that the association between flotillin microdomains and cortical cytoskeleton has important functions during neutrophil migration, in uropod formation, and in the regulation of myosin IIa.

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Direct observation of structure and dynamics during phase separation of an elastomeric protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1701877114 ◽

2017 ◽

Vol 114 (22) ◽

pp. E4408-E4415 ◽

Cited By ~ 78

Author(s):

Sean E. Reichheld ◽

Lisa D. Muiznieks ◽

Fred W. Keeley ◽

Simon Sharpe

Keyword(s):

Phase Separation ◽

Matrix Protein ◽

Molecular Mechanisms ◽

Bulk Water ◽

Extracellular Matrix Protein ◽

Elastic Fibers ◽

Cross Linking ◽

Structure And Dynamics ◽

Chemical Cross Linking

Despite its growing importance in biology and in biomaterials development, liquid–liquid phase separation of proteins remains poorly understood. In particular, the molecular mechanisms underlying simple coacervation of proteins, such as the extracellular matrix protein elastin, have not been reported. Coacervation of the elastin monomer, tropoelastin, in response to heat and salt is a critical step in the assembly of elastic fibers in vivo, preceding chemical cross-linking. Elastin-like polypeptides (ELPs) derived from the tropoelastin sequence have been shown to undergo a similar phase separation, allowing formation of biomaterials that closely mimic the material properties of native elastin. We have used NMR spectroscopy to obtain site-specific structure and dynamics of a self-assembling elastin-like polypeptide along its entire self-assembly pathway, from monomer through coacervation and into a cross-linked elastic material. Our data reveal that elastin-like hydrophobic domains are composed of transient β-turns in a highly dynamic and disordered chain, and that this disorder is retained both after phase separation and in elastic materials. Cross-linking domains are also highly disordered in monomeric and coacervated ELP3 and form stable helices only after chemical cross-linking. Detailed structural analysis combined with dynamic measurements from NMR relaxation and diffusion data provides direct evidence for an entropy-driven mechanism of simple coacervation of a protein in which transient and nonspecific intermolecular hydrophobic contacts are formed by disordered chains, whereas bulk water and salt are excluded.

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Microtubule-entrained kinase activities associated with the cortical cytoskeleton during cytokinesis

Journal of Cell Science ◽

10.1242/jcs.110.12.1373 ◽

1997 ◽

Vol 110 (12) ◽

pp. 1373-1386 ◽

Cited By ~ 2

Author(s):

G.R. Walker ◽

C.B. Shuster ◽

D.R. Burgess

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Protein Phosphorylation ◽

Immunoblot Analysis ◽

Negative Regulator ◽

Mitogen Activated Protein Kinase ◽

Mitotic Apparatus ◽

Cortical Cytoskeleton

Research over the past few years has demonstrated the central role of protein phosphorylation in regulating mitosis and the cell cycle. However, little is known about how the mechanisms regulating the entry into mitosis contribute to the positional and temporal regulation of the actomyosin-based contractile ring formed during cytokinesis. Recent studies implicate p34cdc2 as a negative regulator of myosin II activity, suggesting a link between the mitotic cycle and cytokinesis. In an effort to study the relationship between protein phosphorylation and cytokinesis, we examined the in vivo and in vitro phosphorylation of actin-associated cortical cytoskeletal (CSK) proteins in an isolated model of the sea urchin egg cortex. Examination of cortices derived from eggs or zygotes labeled with 32P-orthophosphate reveals a number of cortex-associated phosphorylated proteins, including polypeptides of 20, 43 and 66 kDa. These three major phosphoproteins are also detected when isolated cortices are incubated with [32P]ATP in vitro, suggesting that the kinases that phosphorylate these substrates are also specifically associated with the cortex. The kinase activities in vivo and in vitro are stimulated by fertilization and display cell cycle-dependent activities. Gel autophosphorylation assays, kinase assays and immunoblot analysis reveal the presence of p34cdc2 as well as members of the mitogen-activated protein kinase family, whose activities in the CSK peak at cell division. Nocodazole, which inhibits microtubule formation and thus blocks cytokinesis, significantly delays the time of peak cortical protein phosphorylation as well as the peak in whole-cell histone H1 kinase activity. These results suggest that a key element regulating cortical contraction during cytokinesis is the timing of protein kinase activities associated with the cortical cytoskeleton that is in turn regulated by the mitotic apparatus.

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