Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells.

The urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein that promotes tissue remodeling, tumor cell adhesion, migration and invasion. uPAR mediates degradation of the extracellular matrix through protease recruitment and enhances cell adhesion, migration and signaling through vitronectin binding and interactions with integrins. Full-length uPAR is released from the cell surface, but the mechanism and significance of uPAR shedding remain obscure. Here we identify transmembrane glycerophosphodiesterase GDE3 as a GPI-specific phospholipase C that cleaves and releases uPAR with consequent loss of function, whereas its homologue GDE2 fails to attack uPAR. GDE3 overexpression depletes uPAR from distinct basolateral membrane domains in breast cancer cells, resulting in a less transformed phenotype, it slows tumor growth in a xenograft model and correlates with prolonged survival in patients. Our results establish GDE3 as a negative regulator of the uPAR signaling network and, furthermore, highlight GPI-anchor hydrolysis as a cell-intrinsic mechanism to alter cell behavior

Sequence-dependent trafficking and activity of GDE2, a GPI-specific phospholipase promoting neuronal differentiation.

GDE2 (also known as GDPD5) is a multispanning membrane phosphodiesterase with phospholipase D-like activity that cleaves select glycosylphosphatidylinositol (GPI)-anchored proteins and thereby promotes neuronal differentiation both in vitro and in vivo GDE2 is a prognostic marker in neuroblastoma, while loss of GDE2 leads to progressive neurodegeneration in mice; however, its regulation remains unclear. Here, we report that, in immature neuronal cells, GDE2 undergoes constitutive endocytosis and travels back along both fast and slow recycling routes. GDE2 trafficking is directed by C-terminal tail sequences that determine the ability of GDE2 to cleave GPI-anchored glypican-6 (GPC6) and induce a neuronal differentiation program. Specifically, we define a GDE2 truncation mutant that shows aberrant recycling and is dysfunctional, whereas a consecutive deletion results in cell-surface retention and gain of GDE2 function, thus uncovering distinctive regulatory sequences. Moreover, we identify a C-terminal leucine residue in a unique motif that is essential for GDE2 internalization. These findings establish a mechanistic link between GDE2 neuronal function and sequence-dependent trafficking, a crucial process gone awry in neurodegenerative diseases.This article has an associated First Person interview with the first author of the paper

Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells

The urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein that promotes tissue remodeling, tumor cell adhesion, migration and invasion. uPAR mediates degradation of the extracellular matrix through protease recruitment and enhances cell adhesion, migration and signaling through vitronectin binding and interactions with integrins. Full-length uPAR is released from the cell surface, but the mechanism and significance of uPAR shedding remain obscure. Here we identify transmembrane glycerophosphodiesterase GDE3 as a GPI-specific phospholipase C that cleaves and releases uPAR with consequent loss of function, whereas its homologue GDE2 fails to attack uPAR. GDE3 overexpression depletes uPAR from distinct basolateral membrane domains in breast cancer cells, resulting in a less transformed phenotype, it slows tumor growth in a xenograft model and correlates with prolonged survival in patients. Our results establish GDE3 as a negative regulator of the uPAR signaling network and, furthermore, highlight GPI-anchor hydrolysis as a cell-intrinsic mechanism to alter cell behavior

Glycerophosphodiesterase GDE2 Promotes Neuroblastoma Differentiation through Glypican Release and Is a Marker of Clinical Outcome

Neuroblastoma is a pediatric embryonal malignancy characterized by impaired neuronal differentiation. A better understanding of neuroblastoma differentiation is essential for developing new therapeutic approaches. GDE2 (encoded by GDPD5) is a six-transmembrane-domain glycerophosphodiesterase that promotes embryonic neurogenesis. We find that high GDPD5 expression is strongly associated with favorable outcome in neuroblastoma. GDE2 induces differentiation of neuroblastoma cells, suppresses cell motility, and opposes RhoA-driven neurite retraction. GDE2 alters the Rac-RhoA activity balance and the expression of multiple differentiation-associated genes. Mechanistically, GDE2 acts by cleaving (in cis) and releasing glycosylphosphatidylinositol-anchored glypican-6, a putative co-receptor. A single point mutation in the ectodomain abolishes GDE2 function. Our results reveal GDE2 as a cell-autonomous inducer of neuroblastoma differentiation with prognostic significance and potential therapeutic value.</p

PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins

Super-resolution microscopy (SRM) allows precise localization of proteins in cellular organelles and structures, including the actin cytoskeleton. Yet sample preparation protocols for SRM are rather anecdotal and still being optimized. Thus, SRM-based imaging of the actin cytoskeleton and associated proteins often remains challenging and poorly reproducible. Here, we show that proper paraformaldehyde (PFA)-based sample preparation preserves the architecture of the actin cytoskeleton almost as faithfully as gold-standard glutaraldehyde fixation. We show that this fixation is essential for proper immuno-based localization of actin-binding and actin-regulatory proteins involved in the formation of lamellipodia and ruffles, such as mDia1, WAVE2 and clathrin heavy chain, and provide detailed guidelines for the execution of our method. In summary, proper PFA-based sample preparation increases the multi-color possibilities and the reproducibility of SRM of the actin cytoskeleton and its associated proteins

Measuring image resolution in optical nanoscopy

Resolution in optical nanoscopy (or super-resolution microscopy) depends on the localization uncertainty and density of single fluorescent labels and on the sample\u27s spatial structure. Currently there is no integral, practical resolution measure that accounts for all factors. We introduce a measure based on Fourier ring correlation (FRC) that can be computed directly from an image. We demonstrate its validity and benefits on two-dimensional (2D) and 3D localization microscopy images of tubulin and actin filaments. Our FRC resolution method makes it possible to compare achieved resolutions in images taken with different nanoscopy methods, to optimize and rank different emitter localization and labeling strategies, to define a stopping criterion for data acquisition, to describe image anisotropy and heterogeneity, and even to estimate the average number of localizations per emitter. Our findings challenge the current focus on obtaining the best localization precision, showing instead how the best image resolution can be achieved as fast as possible

Initiation of lamellipodia and ruffles involves cooperation between mDia1 and the Arp2/3 complex

<p>High resolution figures accompanying the research article entitled: "<strong>Initiation of lamellipodia and ruffles involves cooperation between mDia1 and the Arp2/3 complex</strong>" published in Journal of Cell Science, september 2015 (Isogai et al., 2015; doi: 10.1242/jcs.176768)</p

Isogai et al., JCS 2015 Figure 4

<p><strong>Fig. 4. Full-length mDia1 cooperates with the Arp2/3 complex to form branched actin networks.</strong> (A) Representative Coomassie gels showing purified recombinant wild-type (WT) and mutant (MA) mDia1 (1.5 μg) (top) and WAVE2 (2 μg) (bottom). (B–E) mDia1 promotes both nucleation and elongation of linear actin filaments in the presence of profilin. (B,C) Representative frames extracted from TIRFm time-lapse imaging of actin polymerization at the indicated time and concentration of either mDia1 WT or its MA mutant. Profilin-bound actin (2.5 μM actin+5 μM profilin) was polymerized with the indicated concentration of mDia1 WT (B) or mDia1 MA (C). Scale bars: 10 μm. (D) At least ten filaments (thin lines) were tracked to determine filament length (mean±s.d., thick lines) versus time as described in the Materials and Methods. (E) Elongation rates were derived from D as described in the Materials and Methods. Scatter dot plots show average filament elongation rates. ****P<0.0001 (one-way ANOVA with Bonferroni’s multiple comparison test; n=10–12 filaments). (F) mDia1 accelerates polymerization of branched actin filaments induced by the Arp2/3 complex. The Arp2/3 complex (20 nM) activated by WAVE2 (25 nM) was used to stimulate polymerization of profilin–actin (5 μM and 2.5 μM, respectively), either alone or with increasing concentrations of mDia1 MA. Representative frames extracted from TIRFm time-lapse movies illustrate actin polymerization at the indicated time (s) and concentration of mDia1 MA. Scale bar: 10 μm. (G) mDia1 cooperates with the Arp2/3 complex in making branched actin filaments. The area filled with filaments was quantified as described in the Materials and Methods. A representative dose–response experiment carried out on the same day is shown.</p