Rotenone inhibits axonogenesis via an Lfc/RhoA/ROCK pathway in cultured hippocampal neurons

Rotenone, a broad-spectrum insecticide, piscicide and pesticide, produces a complete and selective suppression of axonogenesis in cultured hippocampal neurons. This effect is associated with an inhibition of actin dynamics through activation of Ras homology member A (RhoA) activity. However, the upstream signaling mechanisms involved in rotenone-induced RhoA activation were unknown. We hypothesized that rotenone might inhibit axon growth by the activation of RhoA/ROCK pathway because of the changes in microtubule (MT) dynamics and the concomitant release of Lfc, a MT-associated Guanine Nucleotide Exchange Factor (GEF) for RhoA. In this study, we demonstrate that rotenone decreases MT stability in morphologically unpolarized neurons. Taxol (3 nM), a drug that stabilizes MT, attenuates the inhibitory effect of rotenone (0.1 μM) on axon formation. Radiometric Forster Resonance Energy Transfer, revealed that this effect is associated with inhibition of rotenone-induced RhoA and ROCK activation. Interestingly, silencing of Lfc, but not of the RhoA GEF ArhGEF1, prevents the inhibitory effect of rotenone on axon formation. Our results suggest that rotenone-induced MT de-stabilization releases Lfc from MT thereby promoting RhoA and ROCK activities and the consequent inhibition of axon growth. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/. (Figure presented.).Fil: Bisbal, Mariano. Universidad Nacional de Córdoba; Argentina. Instituto Universitario de Ciencias Biomédicas de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Remedi, Maria Monica. Instituto Universitario de Ciencias Biomédicas de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Quassollo Infanzon, Gonzalo Emiliano. Instituto Universitario de Ciencias Biomédicas de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Cáceres, Alfredo. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Instituto Universitario de Ciencias Biomédicas de Córdoba; ArgentinaFil: Sanchez, Monica Silvina. Instituto Universitario de Ciencias Biomédicas de Córdoba; Argentina. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin

p27Kip1, an Intrinsically Unstructured Protein with Scaffold Properties

The Cyclin-dependent kinase (CDK) regulator p27Kip1 is a gatekeeper of G1/S transition. It also regulates G2/M progression and cytokinesis completion, via CDK-dependent or -independent mechanisms. Recently, other important p27Kip1 functions have been described, including the regulation of cell motility and migration, the control of cell differentiation program and the activation of apoptosis/autophagy. Several factors modulate p27Kip1 activities, including its level, cellular localization and post-translational modifications. As a matter of fact, the protein is phosphorylated, ubiquitinated, SUMOylated, O-linked N-acetylglicosylated and acetylated on different residues. p27Kip1 belongs to the family of the intrinsically unstructured proteins and thus it is endowed with a large flexibility and numerous interactors, only partially identified. In this review, we look at p27Kip1 properties and ascribe part of its heterogeneous functions to the ability to act as an anchor or scaffold capable to participate in the construction of different platforms for modulating cell response to extracellular signals and allowing adaptation to environmental changes

Integrin and microtubule crosstalk in the regulation of cellular processes

Integrins engage components of the extracellular matrix, and in collaboration with other receptors, regulate signaling cascades that impact cell behavior in part by modulating the cell's cytoskeleton. Integrins have long been known to function together with the actin cytoskeleton to promote cell adhesion, migration, and invasion, and with the intermediate filament cytoskeleton to mediate the strong adhesion needed for the maintenance and integrity of epithelial tissues. Recent studies have shed light on the crosstalk between integrin and the microtubule cytoskeleton. Integrins promote microtubule nucleation, growth, and stabilization at the cell cortex, whereas microtubules regulate integrin activity and remodeling of adhesion sites. Integrin-dependent stabilization of microtubules at the cell cortex is critical to the establishment of apical-basal polarity required for the formation of epithelial tissues. During cell migration, integrin-dependent microtubule stabilization contributes to front-rear polarity, whereas microtubules promote the turnover of integrin-mediated adhesions. This review focuses on this interdependent relationship and its impact on cell behavior and function

Linoleic and oleic acids enhance cell migration by altering the dynamics of microtubules and the remodeling of the actin cytoskeleton at the leading edge

Fatty acids (FA) have a multitude of biological actions on living cells. A target of their action is cell motility, a process of critical importance during cancer cell dissemination. Here, we studied the effect of unsaturated FA on ovarian cancer cell migration in vitro and its role in regulating cytoskeleton structures that are essential for cell motility. Scratch wound assays on human ovary cancer SKOV-3 cell monolayers revealed that low doses (16 μM) of linoleic acid (LA, 18:2 ω6) and oleic acid (OA; 18:1 ω9) promoted migration, while α-linolenic acid (ALA, 18:3 ω3), showed a migration rate similar to that of the control group. Single cell tracking demonstrated that LA and OA-treated cells migrated faster and were more orientated towards the wound closure than control. In vitro addition of those FA resulted in an increased number, length and protrusion speed of filopodia and also in a prominent and dynamic lamellipodia at the cell leading edge. Using time-lapse video-microscopy and FRAP we observed an increase in both the speed and frequency of actin waves associated with more mobile actin and augmented Rac1 activity. We also observed that FA induced microtubule-organizing center (MTOC)-orientation towards the cell front and affected the dynamics of microtubules (MT) in the direction of cell migration. We propose that environmental cues such as OA and LA present in ascitic fluid, should be taken into account as key factors for the regulation of cell migration.Fil: Masner Moratorio, Martin. Instituto Universitario de Ciencias Biomédicas de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Grupo Vinculado Centro de Investigación en Medicina Traslacional Severo R. Amuchástegui - Cimetsa | Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Grupo Vinculado Centro de Investigación en Medicina Traslacional Severo R. Amuchástegui - Cimetsa | Instituto de Investigación Médica Mercedes y Martín Ferreyra. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Grupo Vinculado Centro de Investigación en Medicina Traslacional Severo R. Amuchástegui - Cimetsa; ArgentinaFil: Lujea, Noelia Carolina. Instituto Universitario de Ciencias Biomédicas de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Grupo Vinculado Centro de Investigación en Medicina Traslacional Severo R. Amuchástegui - Cimetsa | Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Grupo Vinculado Centro de Investigación en Medicina Traslacional Severo R. Amuchástegui - Cimetsa | Instituto de Investigación Médica Mercedes y Martín Ferreyra. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Grupo Vinculado Centro de Investigación en Medicina Traslacional Severo R. Amuchástegui - Cimetsa; ArgentinaFil: Bisbal, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Acosta, Cristian Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Kunda, Patricia Elena. Instituto Universitario de Ciencias Biomédicas de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Grupo Vinculado Centro de Investigación en Medicina Traslacional Severo R. Amuchástegui - Cimetsa | Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Grupo Vinculado Centro de Investigación en Medicina Traslacional Severo R. Amuchástegui - Cimetsa | Instituto de Investigación Médica Mercedes y Martín Ferreyra. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Grupo Vinculado Centro de Investigación en Medicina Traslacional Severo R. Amuchástegui - Cimetsa; Argentin

Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture

mTOR kinase is one of the basic links at the crossroad of several signal transduction pathways. De­re­gulated mTOR kinase signaling accompanies the progress of cancer, diabetes, neurodegenerative disorders and aging. Implication of mTOR inhibitor rapamycin decreases migration and invasion of malignant cells, and metastasis formation. However, a precise mechanism of the regulation of cellular locomotion by mTOR kinase is not fully understood. This article focuses on the recent findings that demonstrated a possible role of mTOR kinase in the regulation of cytoskeleton remodeling and cell migration properties. Detailed studies on this non-canonical mTOR function will extend our knowledge about cell migration and metastasis formation and might improve anti-cancer therapeutic approaches.mTOR кіназa є однією з основних ланок, розташованих на перетині кількох шляхів внутрішньоклітинної передачі сигналу. Дерегуляція сигналінгу mTOR кінази супроводжує розвиток онкологічних захворювань, діабету, нейродегенеративних розладів і старіння. Застосування інгібітора mTOR рапаміцина знижує рівень міграції та інвазії злоякісних клітин і утворення метастазів. Однак, точний механізм регуляції рухливості клітин mTOR кіназою повністю не зрозумілий. Дана стаття присвячена останнім дослідженням, які демонструють можливу роль mTOR кінази в регуляції ре моделювання цитоскелету та міграції клітин. Докладні дослідження цієї неканонічної функції mTOR кінази дозволить розширити наші знання про міграцію клітин і утворення метастазів і може привести до поліпшення протиракових терапевтичних підходів.mTOR киназa является одним из основных звеньев, расположенных на пересечении нескольких путей внутриклеточной передачи сигнала. Дерегуляция сигналинга mTOR киназы сопровождает развитие онкологических заболеваний, диабета, нейродегенеративных расстройств и старения. При­менение ингибитора mTOR рапамицина снижает уровень миграции и инвазии злокачественных клеток и образование метастазов. Однако, точный механизм регуляции подвижности клеток mTOR киназы полностью не изучен. Эта статья посвященна последним исследованиям, которые демонстрируют возможную роль mTOR киназы в регуляции ремоделирования цитоскелета и миграции клеток. Под­роб­ные исследования этой неканонической функции mTOR киназы позволят расширить наши знания о миграции клеток и образование метастазов и может привести к улучшению противораковых терапевтических подходов

The role of the monomeric GTPase RhoA in cardiac fibroblasts

Der spezifische Knockdown von RhoA in neonatalen kardialen Rattenfibroblasten führte auf molekularem Level zu einer Reduktion des Myofibroblastenmarkers α-Glattmuskelaktin und zu einem Anstieg im modifizierten acetylierten Tubulin. Auf subzellulärer Ebene konnte ein Verlust von Stressfasern, Aktinstrukturen höherer Ordnung und eine erhöhte Dichte des Golgi-Apparats beobachtet werden. Außerdem waren die Fokaladhäsionen kürzer und zufällig verteilt, was auf einen Verlust der Zellpolarität hinweist. Auf dem zellulären Level erhöhte der Knockdown von RhoA die Zellfläche aber nicht das Volumen. Diese Veränderungen führten zu einer schnelleren Adhäsion unabhängig vom Substrat, eine Reduktion der Migration in 2D und im Gegensatz dazu eine verbesserte Migration durch eine poröse Membran. Außerdem war die mitogene Antwort der Zellen auf einen Serumstimulus stark reduziert. Eine Veränderung in Zellviabilität konnte zudem nicht beobachtet werden. Die Expression und Sekretion des Fibrose-assoziierten Faktors CTGF war in gehungerten Zellen mit einer Reduktion in RhoA Expression signifikant vermindert, was jedoch in der Anwesenheit eines Serumstimulus aufgehoben werden konnte. Auf einer heterogenen multizellulären Ebene verringerte der Knockdown von RhoA die kontraktile Funktion von generierten künstlichen Herzgeweben unter Kalziumstimulation. Dies ging einher mit einer Reduktion der Expression von α-Glattmuskelaktin und Calsequestrin. Durch die Verwendung spezifischer Inhibitoren der Rho-assoziierten Kinase (ROCK) und HDAC6 konnten einige dieser zellulären Veränderungen imitiert und demensprechend einem Effektorprotein zugeordnet werden. Der ROCK Inhibitor Fasudil konnte die morphologischen Veränderungen und die reduzierte Migrationskapazität in Wildtyp-Fibroblasten abbilden, wobei eine Reduktion der Proliferation nach der Verwendung des HDAC6 Inhibitors Tubastatin A beobachtet wurde

A Role for c-Jun Kinase (JNK) Signaling in Glial Engulfment of Degenerating Axons: A Dissertation

The central nervous system (CNS) is composed of two types of cells: neurons that send electrical signals to transmit information throughout the animal and glial cells. Glial cells were long thought to be merely support cells for the neurons; however, recent work has identified many critical roles for these cells during development and in the mature animal. In the CNS, glial cells act as the resident immune cell and they are responsible for the clearance of dead or dying material. After neuronal injury or death, glial cells become reactive, exhibiting dramatic changes in morphology and patterns of gene expression and ultimately engulfing neuronal debris. This rapid clearance of degenerating neuronal material is thought to be crucial for suppression of inflammation and promotion of functional recovery, but molecular pathways mediating these engulfment events remain poorly defined. Drosophila melanogaster is a genetically tractable model system in which to study glial biology. It has been shown that Drosophila glia rapidly respond to axonal injury both morphologically and molecularly and that they ultimately phagocytose the degenerating axonal debris. This glial response to axonal debris requires the engulfment receptor Draper and downstream signaling molecules dCed-6, Shark, and Rac1. However, much remains unknown about the molecular details of this response. In this thesis I show that Drosophila c-Jun kinase (dJNK) signaling is a critical in vivo mediator of glial engulfment activity. In response to axotomy, glial dJNK signals through a cascade involving the upstream MAPKKKs Slipper and TAK1, the MAPKK MKK4, and ultimately the Drosophila AP-1 transcriptional complex composed of JRA and Kayak to initiate glial phagocytosis of degenerating axons. Interestingly, loss of dJNK also blocked injury-induced up-regulation of Draper levels in glia and glial-specific over-expression of Draper was sufficient to rescue phenotypes associated with loss of dJNK signaling. I have identified the dJNK pathway as a novel mediator of glial engulfment activity and show that a primary role for the glial Slipper/Tak1→MKK4→dJNK→dAP-1 signaling cascade is activation of draper expression after axon injury