Characterization of heterologously expressed fibril filaments, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma

Fibril is a constitutive filament forming cytoskeletal protein of unidentified fold, exclusive to members of genus Spiroplasma. It is hypothesized to undergo conformational changes necessary to bring about Spiroplasma motility through changes in body helicity. However, in the absence of a cofactor such as nucleotide that binds to the protein and drives polymerization, the mechanism driving conformational changes in fibril remains unknown. Sodium dodecyl sulphate (SDS) solubilized the fibril filaments and facilitated fibril purification by affinity chromatography. An alternate protocol for obtaining enriched insoluble fibril filaments has been standardized using density gradient centrifugation method. Visualization of purified protein using electron microscopy demonstrated that it forms filament bundles. Probable domain boundaries of fibril protein were identified based on mass spectrometric analysis of proteolytic fragments. Presence of both α-helical and β-sheet signatures in FT-IR measurements suggests that fibril filaments consist of assembly of folded globular domains, and not a β-strand based aggregation similar to amyloid fibrils.

Intrabody-Induced Cell Death by Targeting the T. brucei Cytoskeletal Protein Tb BILBO1

Trypanosoma brucei belongs to a group of important zoonotic parasites. We investigated how these organisms develop their cytoskeleton (the internal skeleton that controls cell shape) and focused on an essential protein (BILBO1) first described in T. brucei .

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

Multimeric cytoskeletal protein complexes orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed, differential detergent fractionation to simultaneously detect protein complexes in hundreds of individual cells. Fractionation occurs by 60 s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Proteins are measured with a ~5-hour immunoassay. Co-detection of cytoskeletal protein complexes in U2OS cells treated with filamentous actin (F-actin) destabilizing Latrunculin A detects a unique subpopulation (~2%) exhibiting downregulated F-actin, but upregulated microtubules. Thus, some cells may upregulate other cytoskeletal complexes to counteract the stress of Latrunculin A treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin. We find heat shock may dysregulate filamentous and globular actin correlation. In this work, our assay overcomes selectivity limitations to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.

PDLIM1: Structure, function and implication in cancer

PDLIM1, a member of the PDZ-LIM family, is a cytoskeletal protein and functions as a platform to form distinct protein complexes, thus participating in multiple physiological processes such as cytoskeleton regulation and synapse formation. Emerging evidence demonstrates that PDLIM1 is dysregualted in a variety of tumors and plays essential roles in tumor initiation and progression. In this review, we summarize the structure and function of PDLIM1, as well as its important roles in human cancers.

FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins

Lipid droplets (LDs) are critical for lipid storage and energy metabolism. LDs form in the endoplasmic reticulum (ER). However, the molecular basis for LD biogenesis remains elusive. Here, we show that fat storage–inducing transmembrane protein 2 (FIT2) interacts with ER tubule-forming proteins Rtn4 and REEP5. The association is mainly transmembrane domain based and stimulated by oleic acid. Depletion of ER tubule-forming proteins decreases the number and size of LDs in cells and Caenorhabditis elegans, mimicking loss of FIT2. Through cytosolic loops, FIT2 binds to cytoskeletal protein septin 7, an interaction that is also required for normal LD biogenesis. Depletion of ER tubule-forming proteins or septins delays nascent LD formation. In addition, FIT2-interacting proteins are up-regulated during adipocyte differentiation, and ER tubule-forming proteins, septin 7, and FIT2 are transiently enriched at LD formation sites. Thus, FIT2-mediated nascent LD biogenesis is facilitated by ER tubule-forming proteins and septins.