Heteromeric Filaments Research Articles

Chlamydia trachomatis Polymorphic Membrane Proteins (Pmps) Form Functional Homomeric and Heteromeric Oligomers.

Chlamydiae are Gram-negative, obligate intracellular bacteria, which infect animals and humans. Adhesion to host cells, the first step in the infection process, is mediated by polymorphic membrane proteins (Pmps). Pmps constitute the largest chlamydial protein family, with 9 members (subdivided into six subtypes) in C. trachomatis and 21 in C. pneumoniae, and are characterized by the presence of multiple copies of GGA(I,L,V) and FxxN motifs. Motif-rich fragments of all nine C. trachomatis Pmps act as adhesins and are essential for infection. As autotransporters, most Pmp proteins are secreted through their β-barrel domain and localize on the surface of the chlamydial cell, where most of them are proteolytically processed. Classical autotransporters are monomeric proteins, which can function as toxins, proteases, lipases and monoadhesive adhesins. Here we show that selected recombinant C. trachomatis Pmp fragments form functional adhesion-competent multimers. They assemble into homomeric and heteromeric filaments, as revealed by non-denaturing gel electrophoresis, size-exclusion chromatography and electron microscopy. Heteromeric filaments reach 2 μm in length, significantly longer than homomeric structures. Filament formation was independent of the number of motifs present in the fragment(s) concerned and their relative affinity for host cells. Our functional studies demonstrated that only adhesion-competent oligomers were able to block a subsequent infection. Pre-loading of infectious chlamydial cells with adhesion-competent Pmp oligomers maintained the subsequent infection, while adhesion-incompetent structures reduced infectivity, presumably by blocking the function of endogenous Pmps. The very large number of possible heteromeric and homomeric Pmp complexes represents a novel mechanism to ensure stable adhesion and possibly host cell immune escape.

Protein Kinase A-Mediated Septin7 Phosphorylation Disrupts Septin Filaments and Ciliogenesis.

Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation.

SEPT12 phosphorylation results in loss of the septin ring/sperm annulus, defective sperm motility and poor male fertility.

Septins are critical for numerous cellular processes through the formation of heteromeric filaments and rings indicating the importance of structural regulators in septin assembly. Several posttranslational modifications (PTMs) mediate the dynamics of septin filaments in yeast. However, little is known about the role of PTMs in regulating mammalian septin assembly, and the in vivo significance of PTMs on mammalian septin assembly and function remains unknown. Here, we showed that SEPT12 was phosphorylated on Ser198 using mass spectrometry, and we generated SEPT12 phosphomimetic knock-in (KI) mice to study its biological significance. The homozygous KI mice displayed poor male fertility due to deformed sperm with defective motility and loss of annulus, a septin-based ring structure. Immunohistochemistry of KI testicular sections suggested that SEPT12 phosphorylation inhibits septin ring assembly during annulus biogenesis. We also observed that SEPT12 was phosphorylated via PKA, and its phosphorylation interfered with SEPT12 polymerization into complexes and filaments. Collectively, our data indicate that SEPT12 phosphorylation inhibits SEPT12 filament formation, leading to loss of the sperm annulus/septin ring and poor male fertility. Thus, we provide the first in vivo genetic evidence characterizing importance of septin phosphorylation in the assembly, cellular function and physiological significance of septins.

Nestin and vimentin intermediate filaments expression in cutaneous melanoma

Nestin, a class VI intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue, is considered as cancer stem cell marker of malignancies of neuroectodermal origin. It may play a role in connecting the components of the cytoskeleton and coordinate changes in cell dynamics. It is well known nestin copolymerizes with class III IF-proteins into heteromeric filaments, mostly vimentin, an intermediate filament overexpressed in various type of cancer. Moreover, nestin contributes to the disassembly of vimentin during mitosis. Vimentin is considered to be an ectodermal, neural and pancreatic progenitor cells marker, and its presence was detected in pancreatic cancer stem-like cells. The cancer stem cell hypothesis suggests that mutated melanocyte stem cells are present in skin as precursors of melanoma cells. Several investigations have provided evidence that the genetic and/or epigenic alterations occurring in the multipotent tissue-specific adult stem cells and/or their early progenies may lead to their malignant transformation into cancer progenitor cells. Melanomagenesis and tumor progression are commonly described as ‘de-differentiation’ processes of transformed, mature melanocytes; recently, it has also been demonstrated that metastatic melanoma cell lines exhibit morphological, phenotypic and functional features of stem cell population. Our recent study suggests that nestin expression in both tumoral and endothelial cells may be considered as an important early prognostic marker in melanoma; on the other hand vimentin overexpression in cancer well correlates with accelerated tumor growth, invasion, and poor prognosis. Based on these considerations, the aim of our study was to investigate the colocalization of nestin and vimentin in primary melanoma by immunofluorescence, and its association with clinico-pathological features and patients survival.

Nestin Modulates Glucocorticoid Receptor Function by Cytoplasmic Anchoring

Nestin is the characteristic intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue. Nestin copolymerizes with class III IF-proteins, mostly vimentin, into heteromeric filaments. Its expression is downregulated with differentiation. Here we show that a strong nestin expression in mouse embryo tissue coincides with a strong accumulation of the glucocorticoid receptor (GR), a key regulator of growth and differentiation in embryonic development. Microscopic studies on cultured cells show an association of GR with IFs composed of vimentin and nestin. Cells lacking nestin, but expressing vimentin, or cells expressing vimentin, but lacking nestin accumulate GR in the nucleus. Completing these networks with an exogenous nestin, respectively an exogenous vimentin restores cytoplasmic anchoring of GR to the IF system. Thus, heteromeric filaments provide the basis for anchoring of GR. The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system. Ligand addition releases GR from IFs and shifts the receptor into the nucleus. Suppression of nestin by specific shRNA abolishes anchoring of GR, induces its accumulation in the nucleus and provokes an irreversible G1/S cell cycle arrest. Suppression of GR prior to that of nestin prevents entry into the arrest. The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR. We hypothesize that expression of nestin is a major determinant in suppression of anti-proliferative activity of GR in undifferentiated tissue and facilitates activation of this growth control in a precise tissue and differentiation dependent manner.

Role of Septin Cytoskeleton in Spine Morphogenesis and Dendrite Development in Neurons

Septins are GTP-binding proteins that polymerize into heteromeric filaments and form microscopic bundles or ring structures in vitro and in vivo. Because of these properties and their ability to associate with membrane, F-actin, and microtubules, septins have been generally regarded as cytoskeletal components [1, 2]. Septins are known to play roles in cytokinesis, in membrane trafficking, and as structural scaffolds; however, their function in neurons is poorly understood. Many members of the septin family, including Septin 7 (Sept7), were found by mass-spectrometry analysis of postsynaptic density (PSD) fractions of the brain [3, 4], suggesting a possible postsynaptic function of septins in neurons. We report that Sept7 is localized at the base of dendritic protrusions and at dendritic branch points in cultured hippocampal neurons--a distribution reminiscent of septin localization in the bud neck of budding yeast. Overexpression of Sept7 increased dendrite branching and the density of dendritic protrusions, whereas RNA interference (RNAi)-mediated knockdown of Sept7 led to reduced dendrite arborization and a greater proportion of immature protrusions. These data suggest that Sept7 is critical for spine morphogenesis and dendrite development during neuronal maturation.