Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay

We have shown previously that the raft-associated proteins flotillin-1 and -2 are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Other proteins such as the adhesion receptor PSGL-1, the actin-membrane linker proteins ezrin/radixin/moesin (ERM) and the signaling enzyme phosphatidylinositol-4-phosphate 5-kinase type Iγ90 (PIPKIγ90) also accumulate in the T-cell uropod. Using the in situ proximity ligation assay (PLA) we now have investigated putative close associations of these proteins in human freshly isolated T-cells before and after chemokine addition. The PLA allows in situ subcellular localization of close proximity of endogenous proteins at single-molecule resolution in fixed cells. It allows detection also of weaker and transient complexes that would not be revealed with co-immunoprecipitation approaches. We previously provided evidence for heterodimer formation of tagged flotillin-1 and -2 in T-cells before and after chemokine addition using fluorescence resonance energy transfer (FRET). We now confirm these findings using PLA for the endogenous flotillins in fixed human T-cells. Moreover, in agreement with the literature, our PLA findings confirm a close association of endogenous PSGL-1 and ERM proteins both in resting and chemokine-activated human T-cells. In addition, we provide novel evidence using the PLA for close associations of endogenous activated ERM proteins with PIPKIγ90 and of endogenous flotillins with PSGL-1 in human T-cells, before and after chemokine addition. Our findings suggest that preformed clusters of these proteins coalesce in the uropod upon cell stimulation

Studies of intercellular Ca2+ signaling and gap-junction coupling in the developing cochlea of mouse models affected by congenital hearing loss

Connexin 26 (Cx26) and connexin 30 (Cx30) form gap junction channels that allow the intercellular diffusion of the Ca2+ mobilizing second messenger IP3. They also form hemichannels that release ATP from the endolymphatic surface of cochlear supporting and epithelial cells. Released ATP in turn activates G-protein coupled P2Y2 and P2Y4 receptors, PLC-dependent generation of IP3, release of Ca2+ from intracellular stores, permitting the regenerative propagation of intercellular Ca2+ signals. In the course of this work, we found that cochlear non-sensory cells of the greater and lesser epithelial ridge (GER and LER, respectively) share the same PLC- and IP3R-dependent signal transduction cascade activated by ATP. In addition, we demonstrated that ATP-dependent Ca2+ signaling activity in cochlear non-sensory cells is spatially graded from the apex to the base of the cochlea during the first postnatal week. Ca2+ signaling under these conditions depends on inositol-1,4,5-trisphosphate generation from phospholipase C (PLC)-dependent hydrolysis of PI(4,5)P(2). Thus we analyzed mice with defective expression of PIPKIγ and found that (i) this enzyme is essential for the acquisition of hearing; (ii) it is primarily responsible for the synthesis of the receptor-regulated PLC-sensitive PI(4,5)P(2) pool in the cell syncytia that supports auditory hair cells and; (iii) spatially graded impairment of the PIP2-IP3-Ca2+ signaling pathway in cochlear non-sensory cells affects the level of gap junction coupling. Vice versa, we found defective gap junction coupling and intercellular IP3-dependent Ca2+ signaling the cochlea of mice with targeted ablation Cx26 or Cx30, as well as in mice knock in for a point mutation (Cx30T5M) associated with human congenital deafness. Altogether, our findings link bidirectionally defective hearing acquisition to Ca2+ signaling impairment and decreased biochemical coupling in the developing cochlea. Transduction of connexin deficient cochlear cultures with a bovine adeno associated virus vectors encoding Cx26 or Cx30 restored protein expression, rescued both gap junction coupling and Ca2+ signaling. Based on this work, we conclude that in vivo connexin gene delivery to the inner ear is a route worth exploring to rescue hearing function in mouse models of deafness and, in future, may lead to the development of therapeutic interventions in humans

On barrier and modified barrier multigrid methods for 3d topology optimization

One of the challenges encountered in optimization of mechanical structures, in particular in what is known as topology optimization, is the size of the problems, which can easily involve millions of variables. A basic example is the minimum compliance formulation of the variable thickness sheet (VTS) problem, which is equivalent to a convex problem. We propose to solve the VTS problem by the Penalty-Barrier Multiplier (PBM) method, introduced by R.\ Polyak and later studied by Ben-Tal and Zibulevsky and others. The most computationally expensive part of the algorithm is the solution of linear systems arising from the Newton method used to minimize a generalized augmented Lagrangian. We use a special structure of the Hessian of this Lagrangian to reduce the size of the linear system and to convert it to a form suitable for a standard multigrid method. This converted system is solved approximately by a multigrid preconditioned MINRES method. The proposed PBM algorithm is compared with the optimality criteria (OC) method and an interior point (IP) method, both using a similar iterative solver setup. We apply all three methods to different loading scenarios. In our experiments, the PBM method clearly outperforms the other methods in terms of computation time required to achieve a certain degree of accuracy

p75 neurotrophin receptor mediates neuronal cell death by activating GIRK channels through phosphatidylinositol 4,5-bisphosphate

The pan neurotrophin receptor p75(NTR) signals programmed cell death both during nervous system development and after neural trauma and disease in the adult. However, the molecular pathways by which death is mediated remain poorly understood. Here, we show that this cell death is initiated by activation of G-protein-coupled inwardly rectifying potassium (GIRK/Kir3) channels and a consequent potassium efflux. Death signals stimulated by neurotrophin-mediated cleavage of p75(NTR) activate GIRK channels through the generation and binding of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2/PIP2] to GIRK channels. Both GIRK channel activity and p75(NTR)-mediated neuronal death are inhibited by sequestration of PtdIns(4,5)P2 and application of GIRK channel inhibitors, whereas pertussis toxin treatment has no effect. Thus, p75(NTR) activates GIRK channels without the need for G(i/o)-proteins. Our results demonstrate a novel mode of activation of GIRK channels, representing an early step in the p75(NTR)-mediated cell death pathway and suggesting a function for these channels during nervous system development

The functional role of PI(4,5)P2 metabolism at septin-enriched plasmalemmal microdomains

The process of cytokinesis ultimately results in the partitioning of a mother cell into two daughter cells. Cytokinesis is initiated through the formation of an actomyosin ring at the equatorial plane of the mother cell, which drives the ingression of a cleavage furrow. At this stage, the scaffolding protein anillin stabilizes the actomyosin ring, and recruits septins. Once the ingression of the cleavage furrow is completed, the furrow membrane anchors to the bundled microtubules of the cytokinetic bridge. This is achieved through the centralspindlin complex, which resides at the nascent midbody organelle. Subsequently, anillin and septins further regulate the maturation of an intercellular bridge (ICB), which will ultimately be resolved by the abscission machinery. The phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] plays a crucial role in mammalian cytokinesis. It is essential for the anillin-dependent stabilization of the actomyosin ring at the cleavage furrow, and it is required for the centralspindlin-dependent tethering of bridge microtubules to the plasma membrane. Nevertheless, the molecular mechanisms orchestrating PI(4,5)P2 synthesis in space and time during cytokinesis have remained elusive so far. In this study we investigated the contribution of type I PIP-kinases (phosphatidylinositol-4-phosphate 5-kinases) to the progression of cytokinesis and unveiled a crucial role for PIPKIy at the ICB. We found that septins interact with a common splice insert of PIPKIy isoforms 3 and 5 (PIPKIy-i3/i5), and that both septins and PIPKIy form a complex with centralspindlin. SiRNA-mediated depletion of PIPKIy-i3/i5 resulted in the loss of septin association with ICB microtubules, scattering of anillin away from the ICB, reduction of centralspindlin at the midbody, and a shorter cytokinetic bridge. Notably, these defects were rescued by wild-type PIPKIy, but not by septin binding-deficient or catalytically inactive mutants. These data support a model wherein septins recruit specific PIPKIy isoforms to the nascent midbody and, at this locale, PIPKIy-i3/i5 synthetize a pool of PI(4,5)P2 required for maintaining anillin, centralspindlin, and septins in place. Super-resolution analysis via ultrastructure-expansion microscopy further confirmed the above-mentioned defects at cytokinetic bridges in cells depleted of PIPKIy-i3/i5. Furthermore, live cell imaging of endogenously tagged eGFP-SEPT6 revealed that the hindered association of septins with microtubules observed at cytokinesis persisted within the daughter cells. Notably, siRNA-mediated depletion of centralspindlin phenocopied the loss of septin association to microtubules in non-mitotic cells, pointing towards a role of centralspindlin in coordinating the translocation of septins to bridge microtubules and, thereby, the inheritance of microtubule-associated septin fibers by daughter cells. Taken together, our findings establish septin-associated PIPKIy isoforms as novel regulators of late cytokinesis and demonstrate that both septins and PIPKIy are essential during midbody assembly and maturation

Type I PIPK-α regulates directed cell migration by modulating Rac1 plasma membrane targeting and activation

PIPKI-α does a job other PIPKI isoforms cannot; it recruits Rac1 to the plasma membrane upon integrin activation, spatially regulating the actin-organizing GTPase during migration

Fuzziness in Protein Interactions: a Historical Perspective

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A role for talin in presynaptic function

Talin, an adaptor between integrin and the actin cytoskeleton at sites of cell adhesion, was recently found to be present at neuronal synapses, where its function remains unknown. Talin interacts with phosphatidylinositol-(4)-phosphate 5-kinase type Iγ, the major phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]–synthesizing enzyme in brain. To gain insight into the synaptic role of talin, we microinjected into the large lamprey axons reagents that compete the talin–PIP kinase interaction and then examined their effects on synaptic structure. A dramatic decrease of synaptic actin and an impairment of clathrin-mediated synaptic vesicle endocytosis were observed. The endocytic defect included an accumulation of clathrin-coated pits with wide necks, as previously observed after perturbing actin at these synapses. Thus, the interaction of PIP kinase with talin in presynaptic compartments provides a mechanism to coordinate PI(4,5)P2 synthesis, actin dynamics, and endocytosis, and further supports a functional link between actin and clathrin-mediated endocytosis

Phosphatidylinositol phosphate kinase type Iγ regulates dynamics of large dense-core vesicle fusion.

Phosphatidylinositol-4,5-bisphosphate was proposed to be an important regulator of large dense-core vesicle exocytosis from neuroendocrine tissues. Here, we have examined the kinetics of secretion in chromaffin cells from mice lacking phosphatidylinositol phosphate kinase type Iγ, the major neuronal phosphatidylinositol-4-phosphate 5-kinase. Absence of this enzyme caused a reduction of the readily releasable vesicle pool and its refilling rate, with a small increase in morphologically docked vesicles, indicating a defect in vesicle priming. Furthermore, amperometry revealed a delay in fusion pore expansion. These results provide direct genetic evidence for a key role of phosphatidylinositol-4,5-bisphosphate synthesis in the regulation of large dense-core vesicle fusion dynamics