Ann P. Wheeler

Rac1 and Rac2 regulate macrophage morphology but are not essential for migration

Rac GTPases are believed to contribute to migration in leukocytes by transducing signals from cell surface receptors to the actin and microtubule cytoskeletons. Mammals have three closely related Rac isoforms, Rac1, Rac2 and Rac3, and it is widely assumed that cell migration requires the activity of these Rac GTPases. We have previously shown that Rac1-null mouse macrophages have altered cell shape and reduced membrane ruffling but normal migration speed. Here we investigate the behaviour of macrophages lacking Rac2 (Rac2–/–) or Rac1 and Rac2 (Rac1/2–/–). Rac2–/– macrophages have reduced F-actin levels and lack podosomes, which are integrin-based adhesion sites, and their migration speed is similar to or slightly slower than wild-type macrophages, depending on the substrate. Unexpectedly, Rac1/2–/– macrophages, which do not express Rac1, Rac2 or Rac3, migrate at a similar speed to wild-type macrophages on a variety of substrates and perform chemotaxis normally, although their morphology and mode of migration is altered. However, Rac1–/– and Rac1/2–/– but not Rac2–/– macrophages are impaired in their ability to invade through Matrigel. Together, these data show that Rac1 and Rac2 have distinct roles in regulating cell morphology, migration and invasion, but are not essential for macrophage migration or chemotaxis.

cGAS surveillance of micronuclei links genome instability to innate immunity

DNA is strictly compartmentalized within the nucleus to prevent autoimmunity; despite this, cyclic GMP–AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.

Toll-like receptor 9 protects non-immune cells from stress by modulating mitochondrial ATP synthesis through the inhibition of SERCA2

Toll‐like receptor 9 (TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro‐inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium‐transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca2+ handling between the SR/ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non‐immune cells—including cardiomyocytes—using the same ligand‐receptor system.

Desmoglein 3 acting as an upstream regulator of Rho GTPases, Rac-1/Cdc42 in the regulation of actin organisation and dynamics

Desmoglein 3 (Dsg3), a member of the desmoglein sub-family, serves as an adhesion molecule in desmosomes. Our previous study showed that overexpression of human Dsg3 in several epithelial lines induces formation of membrane protrusions, a phenotype suggestive of Rho GTPase activation. Here we examined the interaction between Dsg3 and actin in detail and showed that endogenous Dsg3 colocalises and interacts with actin, particularly the junctional actin in a Rac1-dependent manner. Ablation of Rac1 activity by dominant negative Rac1 mutant (N17Rac1) or the Rac1 specific inhibitor (NSC23766) directly disrupts the interaction between Dsg3 and actin. Assembly of the junctional actin at the cell borders is accompanied with enhanced levels of Dsg3, while inhibition of Dsg3 by RNAi results in profound changes in the organisation of actin cytoskeleton. In accordance, overexpression of Dsg3 results in a remarkable increase of Rac1 and Cdc42 activities and to a lesser extent, RhoA. The enhancements in Rho GTPases are accompanied by the pronounced actin-based membrane structures such as lamellipodia and filopodia, enhanced rate of actin turnover and cell polarisation. Together, our results reveal an important novel function for Dsg3 in promoting actin dynamics through regulating Rac1 and Cdc42 activation in epithelial cells.

Desmoglein 3, via an interaction with E-cadherin, is associated with activation of Src.

Background Desmoglein 3 (Dsg3), a desmosomal adhesion protein, is expressed in basal and immediate suprabasal layers of skin and across the entire stratified squamous epithelium of oral mucosa. However, increasing evidence suggests that the role of Dsg3 may involve more than just cell-cell adhesion. Methodology/Principal Findings To determine possible additional roles of Dsg3 during epithelial cell adhesion we used overexpression of full-length human Dsg3 cDNA, and RNAi-mediated knockdown of this molecule in various epithelial cell types. Overexpression of Dsg3 resulted in a reduced level of E-cadherin but a colocalisation with the E-cadherin-catenin complex of the adherens junctions. Concomitantly these transfected cells exhibited marked migratory capacity and the formation of filopodial protrusions. These latter events are consistent with Src activation and, indeed, Src-specific inhibition reversed these phenotypes. Moreover Dsg3 knockdown, which also reversed the decreased level of E-cadherin, partially blocked Src phosphorylation. Conclusions/Significance Our data are consistent with the possibility that Dsg3, as an up-stream regulator of Src activity, helps regulate adherens junction formation.

A Restricted Repertoire of De Novo Mutations in ITPR1 Cause Gillespie Syndrome with Evidence for Dominant-Negative Effect

Gillespie syndrome (GS) is characterized by bilateral iris hypoplasia, congenital hypotonia, non-progressive ataxia, and progressive cerebellar atrophy. Trio-based exome sequencing identified de novo mutations in ITPR1 in three unrelated individuals with GS recruited to the Deciphering Developmental Disorders study. Whole-exome or targeted sequence analysis identified plausible disease-causing ITPR1 mutations in 10/10 additional GS-affected individuals. These ultra-rare protein-altering variants affected only three residues in ITPR1: Glu2094 missense (one de novo, one co-segregating), Gly2539 missense (five de novo, one inheritance uncertain), and Lys2596 in-frame deletion (four de novo). No clinical or radiological differences were evident between individuals with different mutations. ITPR1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. The homo-tetrameric structure has been solved by cryoelectron microscopy. Using estimations of the degree of structural change induced by known recessive- and dominant-negative mutations in other disease-associated multimeric channels, we developed a generalizable computational approach to indicate the likely mutational mechanism. This analysis supports a dominant-negative mechanism for GS variants in ITPR1. In GS-derived lymphoblastoid cell lines (LCLs), the proportion of ITPR1-positive cells using immunofluorescence was significantly higher in mutant than control LCLs, consistent with an abnormality of nuclear calcium signaling feedback control. Super-resolution imaging supports the existence of an ITPR1-lined nucleoplasmic reticulum. Mice with Itpr1 heterozygous null mutations showed no major iris defects. Purkinje cells of the cerebellum appear to be the most sensitive to impaired ITPR1 function in humans. Iris hypoplasia is likely to result from either complete loss of ITPR1 activity or structure-specific disruption of multimeric interactions.

A novel regulatory mechanism links PLCγ1 to PDK1

Summary 3-Phosphoinositide-dependent protein kinase-1 (PDK1) and phospholipase C (PLC)&ggr;1 are two key enzymes in signal transduction that control several intracellular processes. Despite the fact that PLC&ggr;1 has been investigated for several years, the mechanisms of activation of this enzyme are still not completely clear. Similarly, although PDK1 has been mostly investigated for its role in activation of Akt, a crucial enzyme in regulation of several cellular processes, it has become evident recently that the role of PDK1 in physiological and pathological conditions is not limited to Akt activation. Here we demonstrate that PDK1 regulates PLC&ggr;1 activation in a mechanism involving association of the two enzymes and modulation of PLC&ggr;1 tyrosine phosphorylation. We further show that this novel PDK1–PLC&ggr;1 pathway is important for cancer cell invasion. The identification of a PDK1–PLC&ggr;1 pathway reveals the existence of a previously undetected link between two of the most important enzymes in signal transduction. This is likely to have profound consequences for our understanding of several cellular functions that are dependent on phosphoinositides and controlled by PDK1 and PLC&ggr;1.

ROCK1 and ROCK2 regulate epithelial polarisation and geometric cell shape

Cell–cell adhesion and contraction play an essential role in the maintenance of geometric shape and polarisation of epithelial cells. However, the molecular regulation of contraction during cell elongation leading to epithelial polarisation and acquisition of geometric cell shape is not clear.

Synthesis and structure of free-standing germanium quantum dots and their application in live cell imaging

Free-standing Ge quantum dots around 3 nm in size were synthesized using a bench-top colloidal method and suspended in water and ethanol. In the ethanol solution, the photoluminescence of the Ge quantum dots was observed between 650 and 800 nm. Structural and optical properties of these colloidal Ge quantum dots were investigated by utilizing X-ray diffraction, X-ray absorption spectroscopy, Raman spectroscopy, and photoluminescence spectroscopy and transmission electron microscopy. The structure of the as-prepared Ge quantum dots that were found is best described by a core–shell model with a small crystalline core and an amorphous outer shell with a surface that was terminated by hydrogen-related species. As-prepared Ge quantum dots were suspended in cell growth medium, and then loaded into cervical carcinoma (HeLa) cells. The fluorescent microscopy images were then collected using 405 nm, 488 nm, 561 nm and 647 nm wavelengths. We observed that, based on fluorescence measurements, as-prepared Ge quantum dots can remain stable for up to 4 weeks in water. Investigation of toxicity, based on a viability test, of as-prepared uncoated Ge quantum dots in HeLa cells was carried out and compared with the commercial carboxyl coated CdSe/ZnSe quantum dots. The viability tests show that Ge quantum dots are less toxic when compared to commercial carboxyl coated CdSe/ZnS quantum dots.

Super-resolution imaging strategies for cell biologists using a spinning disk microscope.

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging.