Podcasts about limk

¿Podrán las Fuerzas del Cielo convencer a Ian de seguir leyendo luego de un rant de 30 minutos sobre una chispa de Mi3rd4?Igual terminó conmovido... o eso dice.Gustavo se expone a la kriptonita negra, hace comparaciones... y no zafa nadie.Limk al reel que menciono en el episodio:https://www.instagram.com/p/C-NpSW-gNxv/Próxima lectura: All Star Superman de Grant Morrison y Frank Quitely(7-9)⭐⭐⭐⭐⭐⭐Ian Gutierrez - Gus Casals#IanleeCrisis

Committee member,Limerick  Literary Festival, Feb 21st-23rd talking about the legacy of Kate O'Brien in whose honour the festival was set up in 1984. This year is the 40th Limerick Literary Festival and the 50th anniversary of Kate O'Brien's death. Also talking about guest writers who will attend including Claire Keegan, and the award for a first novel by a female writer , worth 2000 euro, sponsored by Bill and Denise Whelan. Opening by Denise Chaila , Limk spoken word artist and singer. Bookings at www.limerickliteraryfestival.com  Saturday Chronicle is kindly sponsored by James M Nash and Co and Derg Kitchen Design   http://dergkitchendesign.ie Originally broadcast on Saturday 27th January 2024 as part of Saturday Chronicle hosted by Tom Hanley and Patricia Anne Moore. Message or what's app the studio on 089 2582647 or email sbcrstudio@gmail.com

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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.30.358861v1?rss=1 Authors: Aihara, S., Fujimoto, S., Sakaguchi, R., Imai, T. Abstract: Developing neurons initially form excessive neurites and then remodel them based on molecular cues and neuronal activity. Developing mitral cells in the olfactory bulb initially extend multiple primary dendrites. They then stabilize single primary dendrites, while eliminating others. However, the mechanisms underlying the selective dendrite remodeling remain elusive. Using CRISPR/Cas9-based knockout screening combined with in utero electroporation, we identified BMPR-2 as a key regulator for the selective dendrite stabilization. Bmpr2 knockout and its rescue experiments show that BMPR-2 inhibits LIMK without ligands and thereby facilitates dendrite destabilization. In contrast, the overexpression of antagonists and agonists indicate that ligand-bound BMPR-2 stabilizes dendrites, most likely by releasing LIMK. Using genetic and FRET imaging experiments, we also demonstrate that free LIMK is activated by NMDARs via Rac1, facilitating dendrite stabilization through F-actin formation. Thus, the selective stabilization of mitral cell dendrites is ensured by concomitant inputs of BMP ligands and neuronal activity. Copy rights belong to original authors. Visit the link for more info

TreatyTalk EP 80 Limk SHC, SFC, Camogie All-Ireland & More by Sporting Limerick

The formation of neuronal networks depends on the proper development and targeting of the neurons within the network. One key challenge during the development of such networks is the correct cross linking of axons and dendrites. Only correct synapse formation between dendrites and axons will allow neurons to contribute to the entire network. Therefore further insights into axon targeting mechanisms will help to understand the underlying developmental processes and contribute to future cures for a number of related diseases. Generally, once a neuron forms an axon, it starts growing towards a certain “target zone”. The underlying axon targeting mechanisms are controlled by a large number of extracellular cues provided by the extracellular matrix and neighboring cells. Depending on the neuron type, axons travel different distances towards their future synaptic partner. During that journey the neurons, more specifically the growth cone, constantly comes into contact with guidance cues. The growth cone symbolizes the forefront of an axon and is responsible to integrate different guidance signals. Depending on their nature, they trigger the local assembly or disassembly of the cytoskeleton and ultimately force the axon to turn into a certain direction. Although different guidance cues activate different signaling pathways, all of these cascades will eventually converge down on the cytoskeleton. These cytoskeletal rearrangements and changes in actin dynamics within the growth cone will promote the turning of the entire axon. In a series of events different guidance cues, attractive and repulsive, will guide the growth cone to its respective target. In this study I used the olfactory system, more specifically the olfactory receptor neurons (ORNs), of Drosophila melanogaster to investigate the mechanisms of axon targeting. The olfactory system of the fruit fly proved to be a very powerful model organism for a number of reasons: First, the number of genetic tools available for Drosophila allows the manipulation of many cellular aspects. Second, ORNs have an extremely stereotyped targeting pattern that proved to be a good system to investigate axon targeting mechanisms. The work presented in this thesis studied the role of the highly conserved actin binding protein Psidin during the development and targeting of ORNs. Herein, I was able to demonstrate that Psidin uses two independent molecular mechanisms to control ORN targeting and survival. To elucidate Psidin’s role in the aforementioned processes, I analyzed two predicted null alleles psidin1 (Brennan et al., 2007) and psidin55D4 (Kim et al., 2011), and one hypomorphic allele psidinIG978 (this study). The new hypomorphic allele psidinIG978 was mapped during this study and found to have a single point mutation within Psidin’s coding region (E320K). The data shown in this study demonstrate that Psidin is required at two different time points during the development of the olfactory system. During ORN development, Psidin is required as non-catalytic part of the N-acetyltransferase complex (NatB) to ensure ORN survival. At later stages during development, Psidin functions as an actin binding protein to regulate actin dynamics to ultimately ensure proper ORN axon targeting. I was able to show for the first time that Psidin’s previously reported function as actin binding protein in oocytes (Kim et al., 2011), is also true for neurons. The loss of Psidin leads to significantly reduced lamellipodia in growth cones of primary neurons in vitro. In agreement with Psidin’s role in actin dynamics is the finding that the parallel removal of the actin stabilizer Tropomyosin rescues the lamellipodia defect in psidin1 primary neurons. This strongly argues for Psidin being an actin destabilizing protein and antagonist of Tropomyosin. In general, psidin1 and psidin55D4 mutant axons showed severe mistargeting defects in vivo – e.g. defasciculation in Or59c and Or42a neurons or ectopic synapse formation in Or47a neurons. However, axons mutant for psidinIG978 displayed a less severe phenotype compared to the null alleles. In agreement with in vitro data, the parallel removal of Tropomyosin rescued the targeting defect in Or59c neurons in vivo. The growth cone and the lamellipodia are both important structures that keep axons responsive towards guidance cues. Therefore the lamellipodia reduction in psidin mutants is likely the cause for the observed targeting defects. Nevertheless, Psidin is required differentially among the ORN classes – the ones that project to dorsolateral or ventromedial glomeruli within the antennal lobe (AL) are more affected than centrally projecting classes. ORN classes that are more affected in psidin mutants have to turn upon entry of the AL. Therefore those classes (dorsolateral and ventromedial) have a higher requirement of Psidin, which has to maintain the lamellipodium, so that the axon can respond to cues in the first place. In addition, I overexpressed different isoforms of LimK and Cofilin to artificially create conditions that favor actin stabilization or destabilization. More generally, conditions that promoted actin destabilization and actin stabilization were able to rescue and aggravate the psidin1 phenotype, respectively. In addition to the targeting defect, psidin1 and psidin55D4 mutants showed a strong reduction in ORN cell numbers. In contrast, cell numbers were not affected in psidinIG978 mutant flies. Again, ORN classes were affected differently – e.g. Or42a neuron number was reduced by 83%, but Or59c number was only reduced by 46%. Indicating Psidin’s function in ORN survival, the expression of the anti-apoptotic protein p35 in psidin mutant neurons selectively rescued the cell number, but failed to rescue the targeting defects. Interestingly, the Psidin/Tropomyosin double mutant showed the opposite effect; here the targeting was rescued, but not the cell number. These findings gave strong indications that Psidin has two independent functions during ORN targeting and development. Psidin is predicted to be the non-catalytic part of the N-acteyltransferase complex B (NatB) in Drosophila (Brennan et al., 2007). Here, Psidin (non-catalytic) forms the NatB-complex together with dNAA20 (catalytic). This complex is thought to acetylate nascent protein chains N-terminally. In this study I demonstrated for the first time that both proteins interact in vivo and in vitro. Indicating that the NatB-complex is involved in ORN survival, the knock-down of dNAA20 in psidinIG978 mutants led to a reduction of ORN cell number that is reminiscent of the cell number in psidin1 or psidin55D4 background. At the same time, the knock-down of dNAA20 had no effect on the targeting of ORNs. Furthermore I was able to show that wild type Psidin and PsidinIG978 interact with dNAA20 at comparable levels in vitro. This is in agreement with the finding that the psidinIG978 allele selectively affects ORN targeting, but not ORN survival. In addition, I was able to map the interaction domain between Psidin and dNAA20. This revealed that the point mutation found in psidinIG978 is just outside of the minimal interaction domain. Deletion of the entire interaction domain led to a complete abolishment of the Psidin/dNAA20 interaction. Furthermore I was able to demonstrate that the interaction of Psidin and dNAA20 is regulated by the phosphorylation of a highly conserved serine residue (S678). Expression of the non-phosphorylatable Psidin isoform (S678A) rescued the targeting and cell number phenotype in vivo. Contrary expression of the phosphomimetic isoform (S678D) only rescued the targeting phenotype, but failed to restore ORN cell number in vivo. In line with this observation is the finding that the S678D isoform is unable to bind dNAA20 in vitro. At the same time the S678A isoform binds dNAA20 at normal levels in vitro. Taken together, the data presented in this work demonstrate that Psidin has two functions during the development and targeting of ORNs using two independent molecular mechanisms: First, during axon targeting Psidin is required as an actin destabilizing molecule and antagonist of Tropomyosin. Psidin maintains the lamellipodia size in growth cones and keeps the cytoskeleton in a dynamic and responsive state. This ensures that growing axons can respond properly to various guidance cues. Second, to ensure ORN survival, Psidin is required as non-catalytic part of the NatB-complex. Here, Psidin interacts with the catalytic subunit dNAA20. The formation of the NatB-complex is regulated by phosphorylation of a conserved serine. In its unphosphorylated state Psidin binds dNAA20 and ensures ORN survival, whereas phosphorylation causes the abolishment of this interaction which results in a reduction of ORN cell number. Concluding, this thesis unambiguously shows that Psidin is required at different time points during the formation of the olfactory system of Drosophila. It utilizes two different pathways to ensure (i) ORN survival as part of the NatB-complex and (ii) ORN targeting as actin binding protein. Due to its strong conservation in higher organisms, the here presented data provide important insights into the function of Psidin’s mammalian homologues.

Die akute Pankreatitis beginnt in den exokrinen Azinuszellen des Pankreas und wird durch verschiedene, bisher nicht vollständig geklärte, intrazelluläre Vorgänge ausgelöst. Das Hormon Cholezystokinin stimuliert Signaltransduktionskaskaden, welche über eine Reorganisation des Aktinzytoskeletts zu einer akuten Organentzündung führen. In dieser Arbeit wurde untersucht, ob ein über das Enzym RhoA vermittelter intrazellulärer Signalweg zu Aktin-bindenden Proteinen im Pankreas diese Reaktion hervorruft und durch Cholezystokinin reguliert werden kann. Die Ergebnisse der vorliegenden Arbeit bringen den Nachweis der Existenz der im Folgenden beschriebenen Signaltransduktionskaskade in exokrinen Azinuszellen: RhoA führt über eine Aktivierung von ROCK II zu einer Phosphorylierung der Zieluntereinheit MYPT1 der Myosinphosphatase und somit zu einer Hemmung des Gesamtenzyms. Dadurch transloziert die sowohl in der Zytosol- als auch in der Zytoskelettfraktion vorkommende, unphosphorylierte Form MYPT1 vollständig ins Zytosol. Die Myosinphosphatase führt zu einer Dephosphorylierung der MLC von Myosin. Die fast vollständig in der Zytoskelettfraktion exprimierte phosphorylierte Form pMLC transloziert im dephosphorylierten Zustand ins Zytosol. Durch die Interaktion mit MYPT1 kann MLC zu einer Aktinmyosinkontraktion und somit zu einer Reorganisation des Aktinzytoskeletts führen. Über alternative Signalwege bewirkt RhoA eine Aktivierung von mDia, welches mittels Profilin zu einer Aktinpolymerisation führt. Über ROCK II wird eine Aktivierung der LIMK durch RhoA vermittelt. Dadurch wird Cofilin vermehrt phosphoryliert, wodurch die Depolymerisation der Aktinfilamente gehemmt wird. Durch eine dosis- und zeitabhängige Stimulation mit physiologischen und supraphysiologischen Dosierungen Cholezystokinin wird der Signalweg über RhoA gehemmt. Dadurch kann eine Kontraktion des Aktinzytoskeletts stattfinden und es zu einer Fusion von Vesikeln und zu einer Inhibierung des regulären Sekretionsmechanismus der Pankreaszellen kommen. Da die Hemmung von Aktin-modulierenden Proteinen eine bedeutende Rolle bei der Organfunktion und Entwicklung der akuten Pankreatitis spielt, trägt diese Arbeit dazu bei, sowohl die physiologischen als auch die pathophysiologischen Vorgänge innerhalb der Azinuszellen näher zu charakterisieren. Dies könnte zu einem besseren Verständnis der dieser Erkrankung zugrundeliegenden Mechanismen führen und somit einen therapeutischen Ansatz bei der akuten Pankreatitis darstellen.

The activation of platelets is a central step during the physiological process of hemostasis and its understanding may lead us to control the pathophysiological process of intra-arterial thrombus formation and vascular occlusion, which can cause acute coronary syndrome and myocardial infarction. One of the important aspects of platelet activation is to understand the dynamic regulation and rearrangement of the cytoskeleton after stimulation. The morphological and functional changes of platelets require a drastic remodeling of the actin cytoskeleton regulated by numerous actin-binding proteins and signaling molecules such as the family of Rho-GTPases. The small GTPase Rho can regulate several aspects of cellular function, predominantly through its downstream effector Rho-kinase. One of the well established Rho-kinase-mediated signaling pathways is the phosphorylation of myosin light chain (MLC) and its counteracting MLC phosphatase. Rho-kinase regulates a second pathway that involves activation of LIM-kinases (LIMKs) and subsequent phosphorylation and inactivation of cofilin, an actin dynamizing protein. Dephosphorylation and activation of cofilin lead to severing and depolymerization of existing actin filaments. The signaling pathway Rho-kinase/LIMKs/cofilin phosphorylation during platelet activation and the question, how the phosphorylation of cofilin affects the actin dynamics underlying platelet activation, has not previously been studied. The physiological agonist thrombin and the pathophysiological relevant agonist lysophosphatidic acid (LPA), which is the main platelet-activating lipid in atherosclerotic plaque, were used as platelet stimuli to address these questions. It was found that the activation of Rho-kinase is important for an increase in F-actin content underlying Ca2+-independent platelet shape change. The activation of Rho-kinase was found to be upstream to secretion and integrin IIbβ3 activation. The rapid activation of Rho-kinase during secretion leads to a further increase in F-actin content as compared to shape change. It was observed that LPA-stimulated dense granule secretion is mainly regulated by Rho-kinase, whereas secretion induced by thrombin was only in part Rho-kinase-dependent. Together, these results show that Rho-kinase regulates the F-actin increase underlying shape change and secretion, but it is not directly involved in aggregation. This study for the first time demonstrates that platelet expresses only LIMK-1 and not LIMK-2. LIMK-1 can be activated by Rho-kinase as well as by p21-activated kinases (PAKs). Our study shows that LIMK-1 activation was mainly Rho-kinase dependent in LPA- and thrombin-stimulated platelets. Although, PAK-1/2 activation was observed during LPA-stimulated platelet shape change, PAKs are unlikely to be involved in LIMK-1 activation in these cells. Like Rho-kinase activation, it was also found that LIMK-1 activation was independent and upstream of integrin IIbβ3 activation. Surprisingly, the activation of LIMK-1 failed to increase cofilin phosphorylation during shape change induced by LPA as well as by thrombin. Inhibition of the Rho-kinase/LIMK-1 pathway unmasked cofilin dephosphorylation suggesting that during shape change the simultaneous activation of a cofilin phosphatase counteracts the effect of LIMK-1 for phosphorylating cofilin. During secretion and aggregation induced by LPA and thrombin, cofilin was rapidly dephosphorylated and subsequently rephosphorylated; the latter phase was due to Rho-kinase/LIMK-1 activation. After stimulation with LPA and thrombin under conditions, where platelet aggregation could not occur, the kinetics of cofilin de- and rephosphorylation were unperturbed indicating their independence of integrin IIbβ3 engagement. Furthermore, the results clearly showed that cofilin dephosphorylation is also independent and upstream of secretion, since the onset of cofilin dephosphorylation was as rapid as secretion in thrombin-stimulated platelets and also occurred in the absence of dense granule secretion in LPA (10 µM)-stimulated platelets. Since the kinetics of cofilin phospho-cycle was similar during secretion and platelet aggregation in LPA- and thrombin-stimulated cells, I propose a general two-step regulatory process for cofilin phospho-cycle underlying primarily secretion, and subsequently platelet aggregation: dephosphorylation by a cofilin phosphatase and then rephosphorylation by the Rho-kinase/LIMK-1 pathway. Our results showing that only dephosphorylated (activated) cofilin binds with F-actin support previous observations that the state of cofilin phosphorylation determines its association with F-actin. The effect of Y-27632 in resting platelets showing a reduction in cofilin phosphorylation, and an increase of F-actin content and cofilin association with F-actin suggested that LIMK-1-mediated cofilin phosphorylation reduces the F-actin content and cofilin association with F-actin in resting platelets. In contrast, during shape change, cofilin that showed no change in its phosphorylation was rapidly associated with the actin cytoskeleton. The maximal cofilin association with actin cytoskeleton occurred before the maximal F-actin increase, suggesting that cofilin association with F-actin might regulate the turnover and actin polymerization during platelet shape change. It is an open question, whether cofilin is locally dephosphorylated before binding to F-actin during shape change. Previous studies in other cells could correlate cofilin dephosphorylation (activation) with the depolymerization of F-actin. However, in our studies cofilin dephosphorylation during the initial phase of thrombin-induced secretion (up to 30 seconds) was associated with a large increase of F-actin and a high amount of cofilin association with F-actin. Cofilin rephosphorylation after 30 seconds did not decrease F-actin content and cofilin association with F-actin. Together, in activated platelets the association of cofilin with F-actin and the F-actin increase do not simply correlate to the cofilin phosphorylation state: it seems to be more complex. It is assumed that the cofilin phosphorylation and actin dynamics are regulated in specific compartments during platelet activation. The rapid cofilin dephosphorylation in platelets was mediated by an okadaic-acid insensitive phosphatase. The activation of the cofilin phosphatase seemed to be regulated at least in part by an increase in intracellular Ca2+ and by PI3-kinase. Cofilin de- and rephosphorylation occurring upstream of secretion and platelet aggregation suggests that the enzymes regulating the cofilin phospho-cycle could be potential targets for the development of anti-thrombotic drugs.

Various stimuli like thrombin induce endothelial cell shape change and stress fiber formation via Rho/Rho-kinase-mediated reorganization of the actin cytoskeleton. LIM-kinases regulate actin cytoskeletal reorganization through phosphorylation of cofilin at Ser3. The LIMK family kinases possess characteristic structural features, consisting of two LIM domains, a PDZ domain and a C-terminal kinase domain. In cell transfection studies it has been shown that LIMK2 is phosphorylated at Thr505 by Rho-kinase thereby activating the enzyme. Recently it has been reported that nuclear LIMKs suppress cyclin D1 expression in a manner independent of cofilin phosphorylation and actin polymerization. In this study, we found that endothelial cells express both LIMK1 and LIMK2. By using live cell imaging, we confirm previous findings that thrombin induces stress fiber formation, ruffle formation and cell contraction. Furthermore, the cell-cell contacts were disrupted and F-actin fibers connecting two cells were broken. Thrombin induced a rapid and sustained Rho-kinase activation and subsequent phosphorylation of LIM-kinase and cofilin. Pretreatment of endothelial cells with the specific Rho-kinase inhibitor Y27632 inhibited MYPT1 phosphorylation, LIM-kinase and cofilin phosphorylation and blocked stress fiber formation in thrombin-stimulated cells. Notably, thrombin induced actin stress fiber formation was abolished in cells transfected with dominant negative LIMK2. LIMK2 was mainly localized in the cytoplasm. By using Leptomycin B (a specific inhibitor of CRM-1 dependent nuclear export) and FRAP and FLIP analysis, we demonstrate that LIMK2 in resting endothelial cells shuttles between the nucleus and cytoplasm. The LIM domains of LIMK2, but not of LIMK1 inhibited its nuclear import thereby keeping LIMK2 mainly in the cytoplasm. Mutational analysis of the unique basic amino acid-rich motif (amino acids 480-503) indicated that this motif regulates the nuclear and nucleolar localization of LIMK2. Activation of PKC in PMA-stimulated endothelial cells stimulated the phosphorylation of LIMK2 at Ser283 and the translocation of LIMK2 and the PDZ-kinase construct of LIMK2 from the nucleus to the cytoplasm. Of the various PKC isoforms, PKC- and PKC- were found to be mainly responsible for Ser283 phosphorylation and the regulation of translocation of LIMK2. Mutational analysis indicated that LIMK2 phosphorylation at Ser283 and Thr494 play a role in the regulation of nucleocytoplasmic shuttling of LIMK2 by PKC. These results show that LIM-kinase activation is mediated by Rho-kinase in stimulated endothelial cells, and that LIM-kinase-mediated cofilin phosphorylation plays an essential role in thrombin-induced stress fiber formation. LIMK2 shuttles between nucleus and cytoplasm in resting endothelial cells. Phosphorylation of LIMK2 at Ser283 and Thr494 by PKC regulates nucleocytoplasmic shuttling and suggests that LIMK2 might also have a function in the nucleus such as the suppression of cyclin D1 expression.