A tent pole twist on membrane ruffles

Macropinocytosis or “cell drinking” involves the elaboration of membrane ruffles that enclose and internalize extracellular fluids. Using lattice light sheet microscopy, Condon et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201804137) reveal the presence of parallel membrane protrusions termed “tent poles” that flank and direct membrane ruffle formation.

Membrane Ruffles Capture C3bi-opsonized Particles in Activated Macrophages

A widespread belief in phagocyte biology is that FcγR-mediated phagocytosis utilizes membrane pseudopods, whereas Mac-1–mediated phagocytosis does not involve elaborate plasma membrane extensions. Here we report that dynamic membrane ruffles in activated macrophages promote binding of C3bi-opsonized particles. We identify these ruffles as components of the macropinocytosis machinery in both PMA- and LPS-stimulated macrophages. C3bi-particle capture is facilitated by enrichment of high-affinity Mac-1 and the integrin-regulating protein talin in membrane ruffles. Membrane ruffle formation and C3bi-particle binding are cytoskeleton dependent events, having a strong requirement for F-actin and microtubules (MTs). MT disruption blunts ruffle formation and PMA- and LPS-induced up-regulation of surface Mac-1 expression. Furthermore, the MT motor, kinesin participates in ruffle formation implicating a requirement for intracellular membrane delivery to active membrane regions during Mac-1–mediated phagocytosis. We observed colocalization of Rab11-positive vesicles with CLIP-170, a MT plus-end binding protein, at sites of particle adherence using TIRF imaging. Rab11 has been implicated in recycling endosome dynamics and mutant Rab11 expression inhibits both membrane ruffle formation and C3bi-sRBC adherence to macrophages. Collectively these findings represent a novel membrane ruffle “capture” mechanism for C3bi-particle binding during Mac-1–mediated phagocytosis. Importantly, this work also demonstrates a strong functional link between integrin activation, macropinocytosis and phagocytosis in macrophages.

Abstract 1449: A Soluble Form Of Ldl Receptor Gene Family Member, Lr11, Is Essential For Angiotensin Ii -induced Smc Migration

Migration of vascular smooth muscle cells (SMCs) from the media to the intima plays a key role in the development of atherosclerosis. Considerable amounts of the soluble form of LR11 (sLR11) are produced by intimal SMCs and enhance migration of SMCs in vitro via upregulation of urokinase-type plasminogen activator receptor (uPAR) expression. Circulating levels of sLR11 are increased in subjects with coronary artery diseases. Here, we show that targeted disruption of the LR11 gene greatly reduces intimal thickening of arteries through attenuation of angiotensin II (Ang II)-induced migration of SMCs. The intimal thickness of femoral arteries after cuff placement was decreased in Lr11 −/− mice. In cultured Lr11 −/− SMCs, AngII-stimulated migration and particularly, attachment, were almost completely abolished. In SMCs, sLR11 caused membrane ruffle formation via activation of focal adhesion kinase (FAK)/extracellular signal-regulated protein kinase (ERK)/Rac1, accompanied by formation of a complex between uPAR and integrin αvβ3. AngII accelerated membrane ruffling through an increase in sLR11-mediated formation of this complex. Overproduction of sLR11 resulted in the reduced sensitivity of AngII-induced activation pathways to inhibition by an AngII type 1 receptor blocker in mice. These results show that AngII-induced SMC migration is mediated by sLR11- induced signal activation for membrane ruffle formation. Our discovery of involvement of LR11 in AngII-induced SMC migration possibly leads to an establishment of novel approach for regulating plaque formation.

Histone Deacetylase 6 Regulates Growth Factor-Induced Actin Remodeling and Endocytosis

ABSTRACT Histone deacetylase 6 (HDAC6) is a cytoplasmic deacetylase that uniquely catalyzes α-tubulin deacetylation and promotes cell motility. However, the mechanism underlying HDAC6-dependent cell migration and the role for microtubule acetylation in motility are not known. Here we show that HDAC6-induced global microtubule deacetylation was not sufficient to stimulate cell migration. Unexpectedly, in response to growth factor stimulation, HDAC6 underwent rapid translocation to actin-enriched membrane ruffles and subsequently became associated with macropinosomes, the vesicles for fluid-phase endocytosis. Supporting the importance of these associations, membrane ruffle formation, macropinocytosis, and cell migration were all impaired in HDAC6-deficient cells. Conversely, elevated HDAC6 levels promoted membrane ruffle formation with a concomitant increase in macropinocytosis and motility. In search for an HDAC6 target, we found that heat shock protein 90 (Hsp90), another prominent substrate of HDAC6, was also recruited to membrane ruffles and macropinosomes. Significantly, inhibition of Hsp90 activity suppressed membrane ruffling and cell migration, while expression of an acetylation-resistant Hsp90 mutant promoted ruffle formation. Our results uncover a surprising role for HDAC6 in actin remodeling-dependent processes and identify the actin cytoskeleton as an important target of HDAC6-regulated protein deacetylation.

Characterization of Pseudomonas aeruginosa Exoenzyme S as a Bifunctional Enzyme in J774A.1 Macrophages

ABSTRACT Pseudomonas aeruginosa exoenzyme S (ExoS) is a type III secretion (TTS) effector, which includes both a GTPase-activating protein (GAP) activity toward the Rho family of low-molecular-weight G (LMWG) proteins and an ADP-ribosyltransferase (ADPRT) activity that targets LMWG proteins in the Ras, Rab, and Rho families. The coordinate function of both activities of ExoS in J774A.1 macrophages was assessed by using P. aeruginosa strains expressing and translocating wild-type ExoS or ExoS defective in GAP and/or ADPRT activity. Distinct and coordinated functions were identified for both domains. The GAP activity was required for the antiphagocytic effect of ExoS and was linked to interference of lamellopodium and membrane ruffle formation. Alternatively, the ADPRT activity of ExoS altered cellular adherence and morphology and was linked to effects on filopodium formation. The cellular mechanism of ExoS GAP activity included an inactivation of Rac1 function, as determined in p21-activated kinase 1-glutathione S-transferase (GST) pull-down assays. The ADPRT activity of ExoS targeted Ras and RalA but not Rab or Rho proteins, and Ral binding protein 1-GST pull-down assays identified an effect of ExoS ADPRT activity on RalA activation. The results from these studies confirm the bifunctional nature of ExoS activity within macrophages when translocated by TTS.

SKIP Negatively Regulates Insulin-Induced GLUT4 Translocation and Membrane Ruffle Formation

ABSTRACT Skeletal muscle and kidney enriched inositol phosphatase (SKIP) is an inositol polyphosphate 5-phosphatase that hydrolyzes phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to downregulate intracellular levels. In this study, we show that SKIP inhibits phosphoinositide 3-kinase signaling in insulin-stimulated CHO cells. Ectopic expression of SKIP did not inhibit insulin-induced PI(3,4,5)P3 generation but did rapidly decrease insulin-induced intracellular PI(3,4,5)P3 levels compared with those in control cells. Further, insulin-induced phosphorylation of some downstream targets such as Akt and p70 S6 kinase was markedly inhibited by the ectopic expression of SKIP, whereas phosphorylation of mitogen-activated protein kinase was not. In contrast, downregulation of intracellular SKIP levels by antisense oligonucleotides dramatically enhanced Akt (protein kinase B) phosphorylation in response to insulin, suggesting that endogenous SKIP downregulates insulin signaling. SKIP also markedly inhibited GLUT4 translocation and membrane ruffle formation. We conclude that SKIP preferentially regulates glucose transport and actin cytoskeletal rearrangement among a variety of PI(3,4,5)P3 downstream events.