Tuning spherical cells into kinking helices in wall-less bacteria

In bacteria, cell shape is determined and maintained through a complex interplay between the peptidoglycan cell wall and cytoplasmic filaments made of polymerized MreB. Spiroplasma species, members of the Mollicutes class, challenge this general understanding because they are characterized by a helical cell shape and motility without a cell wall. This specificity is thought to rely on five MreB isoforms and a specific fibril protein. In this study, combinations of these five MreBs and of the fibril from Spiroplasma citri were expressed in another Mollicutes, Mycoplasma capricolum. Mycoplasma cells that were initially pleomorphic, mostly spherical, turned into helices when MreBs and fibrils were expressed in this heterologous host. The fibril protein was essential neither for helicity nor for cell movements. The isoform MreB5 had a special role as it was sufficient to confer helicity and motility to the mycoplasma cells. Cryo-electron microscopy confirmed the association of MreBs and fibril-based cytoskeleton with the plasma membrane, suggesting a direct effect on the membrane curvature. Finally, the heterologous expression of these proteins, MreBs and fibril, made it possible to reproduce the kink-like motility of spiroplasmas without providing the ability of cell movement in liquid broth. We suggest that other Spiroplasma components, not yet identified, are required for swimming, a hypothesis that could be evaluated in future studies using the same model.

Keratin 7 Is a Constituent of the Keratin Network in Mouse Pancreatic Islets and Is Upregulated in Experimental Diabetes

Keratin (K) 7 is an intermediate filament protein expressed in ducts and glands of simple epithelial organs and in urothelial tissues. In the pancreas, K7 is expressed in exocrine ducts, and apico-laterally in acinar cells. Here, we report K7 expression with K8 and K18 in the endocrine islets of Langerhans in mice. K7 filament formation in islet and MIN6 β-cells is dependent on the presence and levels of K18. K18-knockout (K18‒/‒) mice have undetectable islet K7 and K8 proteins, while K7 and K18 are downregulated in K8‒/‒ islets. K7, akin to F-actin, is concentrated at the apical vertex of β-cells in wild-type mice and along the lateral membrane, in addition to forming a fine cytoplasmic network. In K8‒/‒ β-cells, apical K7 remains, but lateral keratin bundles are displaced and cytoplasmic filaments are scarce. Islet K7, rather than K8, is increased in K18 over-expressing mice and the K18-R90C mutation disrupts K7 filaments in mouse β-cells and in MIN6 cells. Notably, islet K7 filament networks significantly increase and expand in the perinuclear regions when examined in the streptozotocin diabetes model. Hence, K7 represents a significant component of the murine islet keratin network and becomes markedly upregulated during experimental diabetes.

RanBP2/Nup358 enhances miRNA activity by sumoylating Argonautes

Mutations in RanBP2 (also known as Nup358), one of the main components of the cytoplasmic filaments of the nuclear pore complex, contribute to the overproduction of acute necrotizing encephalopathy (ANE1)-associated cytokines. Here we report that RanBP2 represses the translation of the interleukin 6 (IL6) mRNA, which encodes a cytokine that is aberrantly up-regulated in ANE1. Our data indicates that soon after its production, the IL6 messenger ribonucleoprotein (mRNP) recruits Argonautes bound to let-7 microRNA. After this mRNP is exported to the cytosol, RanBP2 sumoylates mRNP-associated Argonautes, thereby stabilizing them and enforcing mRNA silencing. Collectively, these results support a model whereby RanBP2 promotes an mRNP remodelling event that is critical for the miRNA-mediated suppression of clinically relevant mRNAs, such as IL6.

Into the basket and beyond: the journey of mRNA through the nuclear pore complex

The genetic information encoded in nuclear mRNA destined to reach the cytoplasm requires the interaction of the mRNA molecule with the nuclear pore complex (NPC) for the process of mRNA export. Numerous proteins have important roles in the transport of mRNA out of the nucleus. The NPC embedded in the nuclear envelope is the port of exit for mRNA and is composed of ∼30 unique proteins, nucleoporins, forming the distinct structures of the nuclear basket, the pore channel and cytoplasmic filaments. Together, they serve as a rather stationary complex engaged in mRNA export, while a variety of soluble protein factors dynamically assemble on the mRNA and mediate the interactions of the mRNA with the NPC. mRNA export factors are recruited to and dissociate from the mRNA at the site of transcription on the gene, during the journey through the nucleoplasm and at the nuclear pore at the final stages of export. In this review, we present the current knowledge derived from biochemical, molecular, structural and imaging studies, to develop a high-resolution picture of the many events that culminate in the successful passage of the mRNA out of the nucleus.

RanBP2/Nup358 enhances miRNA activity by sumoylating and stabilizing Argonaute 1

SUMMARYRanBP2/Nup358 is one of the main components of the cytoplasmic filaments of the nuclear pore complex. It has been speculated that RanBP2, which has an E3 SUMO-ligase domain, may alter the composition of messenger ribonucleoprotein (mRNP) complexes as they emerge from the nuclear pore and thus regulate the ultimate fate of the mRNA in the cytoplasm. Four separate missense mutations in RanBP2 cause Acute Necrotizing Encephalopathy 1 (ANE1), which manifests as a sharp rise in cytokine production after common viral infections such as influenza and parainfluenza. However, how RanBP2 and its ANE1-associated mutations affect cytokine production is not well understood. Here we report that RanBP2 represses the translation of the interleukin-6 (IL6) mRNA, which encodes a cytokine that is aberrantly up-regulated in ANE1. Our data indicates that soon after its production, the IL6 mRNP recruits the RNA-induced silencing complex (RISC) bound to Let7 miRNA. After this mRNP is exported, RanBP2 sumoylates the RISC-component AGO1, thereby stabilizing it and enforcing mRNA silencing. Collectively, these results support a model whereby RanBP2 promotes an mRNP remodelling event that is critical for the miRNA-mediated suppression of clinically relevant mRNAs, such as IL6.

Rapid isolation of functionally intact nuclei from the yeast Saccharomyces

ABSTRACTMost available methods for nuclear isolation entail lengthy procedures that are difficult to master and generally emphasize yield and enrichment over nuclear preservation, thus limiting their utility for further studies. Here we demonstrate a novel and robust method to rapidly isolate well-preserved yeast nuclei. The method can be easily adapted to multiple preparation scales depending on experimental need and it can readily be performed on multiple samples by a single researcher in one day. We show that the nuclei fraction is strongly enriched and that the resulting nuclei are free from contaminating endoplasmatic reticulum and other cell debris. EM studies show that preservation of nuclear morphology is exquisite, making it possible to study peripheral nuclear pore components such as the cytoplasmic filaments and the basket, whose structure is generally difficult to maintain ex vivo. In addition, incubation of isolated nuclei with bulk transport substrates of different sizes and with import cargo indicates that the nuclear envelope is intact and nuclear pores retain their capacity to bind transport substrates. Our results suggest that this preparation procedure will greatly facilitate studies of the yeast nucleus which have been difficult to establish and to multiplex to date.

Neuronal Wiskott-Aldrich syndrome protein (N-WASP) is critical for formation of α-smooth muscle actin filaments during myofibroblast differentiation

Myofibroblasts are implicated in pathological stromal responses associated with lung fibrosis. One prominent phenotypic marker of fully differentiated myofibroblasts is the polymerized, thick cytoplasmic filaments containing newly synthesized α-smooth muscle actin (α-SMA). These α-SMA-containing cytoplasmic filaments are important for myofibroblast contractility during tissue remodeling. However, the molecular mechanisms regulating the formation and maturation of α-SMA-containing filaments have not been defined. This study demonstrates a critical role for neuronal Wiskott-Aldrich syndrome protein (N-WASP) in regulating the formation of α-SMA-containing cytoplasmic filaments during myofibroblast differentiation and in myofibroblast contractility. Focal adhesion kinase (FAK) is activated by transforming growth factor-β1 (TGF-β1) and is required for phosphorylation of tyrosine residue 256 (Y256) of N-WASP. Phosphorylation of Y256 of N-WASP is essential for TGF-β1-induced formation of α-SMA-containing cytoplasmic filaments in primary human lung fibroblasts. In addition, we demonstrate that actin-related protein (Arp) 2/3 complex is downstream of N-WASP and mediates the maturation of α-SMA-containing cytoplasmic filaments. Together, this study supports a critical role of N-WASP in integrating FAK and Arp2/3 signaling to mediate formation of α-SMA-containing cytoplasmic filaments during myofibroblast differentiation and maturation.

Cryo-Electron Tomography Elucidates the Molecular Architecture of Treponema pallidum, the Syphilis Spirochete

ABSTRACT Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure's fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete's complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator-P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG.