- Browse by Subject
Browsing by Subject "Actin"
Now showing 1 - 10 of 14
Results Per Page
Sort Options
Item The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)-mediated glucose uptake into skeletal muscle cells(American Society for Biochemistry and Molecular Biology, 2017-11-17) Tunduguru, Ragadeepthi; Zhang, Jing; Aslamy, Arianne; Salunkhe, Vishal A.; Brozinick, Joseph T.; Elmendorf, Jeffrey S.; Thurmond, Debbie C.; Biochemistry and Molecular Biology, School of MedicineDefects in translocation of the glucose transporter GLUT4 are associated with peripheral insulin resistance, preclinical diabetes, and progression to type 2 diabetes. GLUT4 recruitment to the plasma membrane of skeletal muscle cells requires F-actin remodeling. Insulin signaling in muscle requires p21-activated kinase-1 (PAK1), whose downstream signaling triggers actin remodeling, which promotes GLUT4 vesicle translocation and glucose uptake into skeletal muscle cells. Actin remodeling is a cyclic process, and although PAK1 is known to initiate changes to the cortical actin-binding protein cofilin to stimulate the depolymerizing arm of the cycle, how PAK1 might trigger the polymerizing arm of the cycle remains unresolved. Toward this, we investigated whether PAK1 contributes to the mechanisms involving the actin-binding and -polymerizing proteins neural Wiskott-Aldrich syndrome protein (N-WASP), cortactin, and ARP2/3 subunits. We found that the actin-polymerizing ARP2/3 subunit p41ARC is a PAK1 substrate in skeletal muscle cells. Moreover, co-immunoprecipitation experiments revealed that insulin stimulates p41ARC phosphorylation and increases its association with N-WASP coordinately with the associations of N-WASP with cortactin and actin. Importantly, all of these associations were ablated by the PAK inhibitor IPA3, suggesting that PAK1 activation lies upstream of these actin-polymerizing complexes. Using the N-WASP inhibitor wiskostatin, we further demonstrated that N-WASP is required for localized F-actin polymerization, GLUT4 vesicle translocation, and glucose uptake. These results expand the model of insulin-stimulated glucose uptake in skeletal muscle cells by implicating p41ARC as a new component of the insulin-signaling cascade and connecting PAK1 signaling to N-WASP-cortactin-mediated actin polymerization and GLUT4 vesicle translocation.Item ADF/Cofilin Activation Regulates Actin Polymerization and Tension Development in Canine Tracheal Smooth Muscle(2009-09-03T15:28:09Z) Zhao, Rong; Gunst, Susan J.; Atkinson, Simon J.; Elmendorf, Jeffrey S.; Sturek, Michael S.The contractile activation of airway smooth muscle tissues stimulates actin polymerization and the inhibition of actin polymerization inhibits tension development. Actin depolymerizing factor (ADF) and cofilin are members of a family of actin–binding proteins that mediate the severing of F–actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of airway smooth was evaluated in intact canine tracheal smooth muscle tissues. Two–dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated (inactivated). Phospho–ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho–cofilin mimetic (cofilin S3E), but not WT cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh–induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh–induced dephosphorylation of ADF/cofilin required the Ca2+–dependent activation of calcineurin (PP2B). Expression of Slingshot (SSH) inactive phosphatase (C393S) decreased force development and cofilin dephosphorylation. Activation of ADF/cofilin was also required for the relaxation of tracheal muscle tissues induced by forskolin and isoproterenol. Cofilin activation in response to forskolin was not Ca2+–dependent and was not inhibited by calcineurin inhibitors, suggesting it was regulated by a different mechanism. Cofilin activation is required for actin dynamics and tension development in response to the contractile stimulation of tracheal smooth muscle and is regulated by both contractile and relaxing stimuli. These concepts are critical to understanding the mechanisms of smooth muscle contraction and relaxation, which may provide novel targets for therapeutic intervention in the treatment of abnormal airway responsiveness.Item Cross-linked actin networks (CLANs) affect stiffness and/or actin dynamics in transgenic transformed and primary human trabecular meshwork cells(Elsevier, 2022) Peng, Michael; Rayana, Naga Pradeep; Dai, Jiannong; Sugali, Chenna Kesavulu; Baidouri, Hasna; Suresh, Ayush; Raghunathan, Vijay Krishna; Mao, Weiming; Ophthalmology, School of MedicineCross-linked actin networks (CLANs) in trabecular meshwork (TM) cells may contribute to increased IOP by altering TM cell function and stiffness. However, there is a lack of direct evidence. Here, we developed transformed TM cells that form spontaneous fluorescently labeled CLANs. The stable cells were constructed by transducing transformed glaucomatous TM (GTM3) cells with the pLenti-LifeAct-EGFP-BlastR lentiviral vector and selection with blastcidin. The stiffness of the GTM3-LifeAct-GFP cells were studied using atomic force microscopy. Elastic moduli of CLANs in primary human TM cells treated with/without dexamethasone/TGFβ2 were also measured to validate findings in GTM3-LifeAct-GFP cells. Live-cell imaging was performed on GTM3-LifeAct-GFP cells treated with 1μM latrunculin B or pHrodo bioparticles to determine actin stability and phagocytosis, respectively. The GTM3-LifeAct-GFP cells formed spontaneous CLANs without the induction of TGFβ2 or dexamethasone. The CLAN containing cells showed elevated cell stiffness, resistance to latrunculin B-induced actin depolymerization, as well as compromised phagocytosis, compared to the cells without CLANs. Primary human TM cells with dexamethasone or TGFβ2-induced CLANs were also stiffer and less phagocytic. The GTM3-LifeAct-GFP cells are a novel tool for studying the mechanobiology and pathology of CLANs in the TM. Initial characterization of these cells showed that CLANs contribute to at least some glaucomatous phenotypes of TM cells.Item DISHEVELLED-ASSOCIATED ACTIVATOR OF MORPHOGENESIS 1 (DAAM1) IS REQUIRED FOR HEART MORPHOGENESIS(2010-02-02T19:55:35Z) Li, Deqiang; Shou, Weinian; Field, Loren J.; Payne, R. Mark; Zhang, XinDishevelled-associated activator of morphogenesis 1 (Daam1), a member of the formin protein family, has been implicated in the non-canonical Wnt mediated Planar Cell Polarity (PCP) signaling pathway. Although the studies in Drosophila Daam1 and Xenopus Daam1 generated inconsistent conclusions regarding the function of Daam1, the biological function of mammalian Daam1 was not evaluated. In this study, we used a mouse promoter trap technology to create Daam1 deficient mice to analyze the role of Daam1 in embryonic development and organogenesis. Daam1 is highly expressed in the developing heart. The majority of Daam1 mutant mice died between embryonic day 14.5 and birth, exhibiting a variety of heart defects, which include ventricular noncompaction, ventricular septal defects, and double outlet right ventricle. About 10% mutant mice survive to adulthood, and these survivors do not show significantly compromised heart function based on echocardiographic analyses. However, all of these mutant survivors have ventricular noncompaction with a range of severities. A conditional rescue experiment using a cardiac specific Cre mouse line, Nkx2-5Cre, confirmed that the cardiac defects are the primary cause of death in Daam1 mutants. Both in vivo and ex vivo analyses revealed that Daam1 is essential for regulating non-sarcomeric filamentous actin assembly in cardiomyocytes, which likely contributes to cardiac morphogenesis and ventricular wall maturation. Biochemical studies further suggested that Daam1 is not a key signaling component in regulating the activation of small GTPases, such as RhoA, Rac1 and Cdc42. In conclusion, our studies demonstrated that Daam1 is essential for cardiac morphogenesis likely through its regulation of cytoskeletal architecture in the developing cardiomyocytes.Item Effect of Shear Stress on RhoA Activities and Cytoskeletal Organization in Chondrocytes(2013-09-05) Wan, Qiaoqiao; Na, Sungsoo; Li, Jiliang; Yokota, HirokiMechanical force environment is a major factor that influences cellular homeostasis and remodeling. The prevailing wisdom in this field demonstrated that a threshold of mechanical forces or deformation was required to affect cell signaling. However, by using a fluorescence resonance energy transfer (FRET)-based approach, we found that C28/I2 chondrocytes exhibited an increase in RhoA activities in response to high shear stress (10 or 20 dyn/cm2), while they showed a decrease in their RhoA activities to intermediate shear stress at 5 dyn/cm2. No changes were observed under low shear stress (2 dyn/ cm2). The observed two-level switch of RhoA activities was closely linked to the shear stress-induced alterations in actin cytoskeleton and traction forces. In the presence of constitutively active RhoA (RhoA-V14), intermediate shear stress suppressed RhoA activities, while high shear stress failed to activate them. Collectively, these results herein suggest that intensities of shear stress are critical in differential activation and inhibition of RhoA activities in chondrocytes.Item The Effects of Chromium on Skeletal Muscle Membrane/Cytoskeletal Parameters and Insulin Sensitivity(2012-07-03) Hoffman, Nolan John; Elmendorf, Jeffrey S.; Considine, Robert V.; Morral, Nuria; Pavalko, Fredrick M.A recent review of randomized controlled trials found that trivalent chromium (Cr3+) supplementation significantly improved glycemia among patients with diabetes, consistent with a long-standing appreciation that this micronutrient optimizes carbohydrate metabolism. Nevertheless, a clear limitation in the current evidence is a lack of understanding of Cr3+ action. We tested if increased AMP-activated protein kinase (AMPK) activity, previously observed in Cr3+-treated cells or tissues from Cr3+-supplemented animals, mediates improved glucose transport regulation under insulin-resistant hyperinsulinemic conditions. In L6 myotubes stably expressing the glucose transporter GLUT4 carrying an exofacial myc-epitope tag, acute insulin stimulation increased GLUT4myc translocation by 69% and glucose uptake by 97%. In contrast, the hyperinsulinemic state impaired insulin stimulation of these processes. Consistent with Cr3+’s beneficial effect on glycemic status, chromium picolinate (CrPic) restored insulin’s ability to fully regulate GLUT4myc translocation and glucose transport. Insulin-resistant myotubes did not display impaired insulin signaling, nor did CrPic amplify insulin signaling. However, CrPic normalized elevated membrane cholesterol that impaired cortical filamentous actin (F-actin) structure. Mechanistically, data support that CrPic lowered membrane cholesterol via AMPK. Consistent with this data, siRNA-mediated AMPK silencing blocked CrPic’s beneficial effects on GLUT4 and glucose transport regulation. Furthermore, the AMPK agonist 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleoside (AICAR) protected against hyperinsulinemia-induced membrane/cytoskeletal defects and GLUT4 dysregulation. To next test Cr3+ action in vivo, we utilized obesity-prone C57Bl/6J mice fed a low fat (LF) or high fat (HF) diet for eight weeks without or with CrPic supplementation administered in the drinking water (8 µg/kg/day). HF feeding increased body weight beginning four weeks after diet intervention regardless of CrPic supplementation and was independent of changes in food consumption. Early CrPic supplementation during a five week acclimation period protected against glucose intolerance induced by the subsequent eight weeks of HF feeding. As observed in other insulin-resistant animal models, skeletal muscle from HF-fed mice displayed membrane cholesterol accrual and loss of F-actin. Skeletal muscle from CrPic-supplemented HF-fed mice showed increased AMPK activity and protection against membrane cholesterol accrual and F-actin loss. Together these data suggest a mechanism by which Cr3+ may positively impact glycemic status, thereby stressing a plausible beneficial action of Cr3+ in glucose homeostasis.Item Exercise training prevents skeletal muscle plasma membrane cholesterol accumulation, cortical actin filament loss, and insulin resistance in C57BL/6J mice fed a western‐style high‐fat diet(Wiley, 2017-08-15) Ambery, Ashley G.; Tackett, Lixuan; Penque, Brent A.; Brozinick, Joseph T.; Elmendorf, Jeffrey S.; Cellular and Integrative Physiology, School of MedicineInsulin action and glucose disposal are enhanced by exercise, yet the mechanisms involved remain imperfectly understood. While the causes of skeletal muscle insulin resistance also remain poorly understood, new evidence suggest excess plasma membrane (PM) cholesterol may contribute by damaging the cortical filamentous actin (F‐actin) structure essential for GLUT4 glucose transporter redistribution to the PM upon insulin stimulation. Here, we investigated whether PM cholesterol toxicity was mitigated by exercise. Male C57BL/6J mice were placed on low‐fat (LF, 10% kCal) or high‐fat (HF, 45% kCal) diets for a total of 8 weeks. During the last 3 weeks of this LF/HF diet intervention, all mice were familiarized with a treadmill for 1 week and then either sham‐exercised (0 m/min, 10% grade, 50 min) or exercised (13.5 m/min, 10% grade, 50 min) daily for 2 weeks. HF‐feeding induced a significant gain in body mass by 3 weeks. Sham or chronic exercise did not affect food consumption, water intake, or body mass gain. Prior to sham and chronic exercise, “pre‐intervention” glucose tolerance tests were performed on all animals and demonstrated that HF‐fed mice were glucose intolerant. While sham exercise did not affect glucose tolerance in the LF or HF mice, exercised mice showed an improvement in glucose tolerance. Muscle from sham‐exercised HF‐fed mice showed a significant increase in PM cholesterol, loss of cortical F‐actin, and decrease in insulin‐stimulated glucose transport compared to sham‐exercised LF‐fed mice. These HF‐fed skeletal muscle membrane/cytoskeletal abnormalities and insulin resistance were improved in exercised mice. These data reveal a new therapeutic aspect of exercise being regulation of skeletal muscle PM cholesterol homeostasis. Further studies on this mechanism of insulin resistance and the benefits of exercise on its prevention are needed.Item Mechanisms of hexosamine-induced cholesterol accumulation and therapeutic actions of chromium(2014-01-03) Penque, Brent A.; Elmendorf, Jeffrey S.; Atkinson, Simon; Considine, Robert V.; Evans-Molina, Carmella; Kempson, Stephen A.Excess caloric intake and/or obesity currently remain the largest predisposing risk factors for the development of type 2 diabetes. Discerning the cellular and molecular mechanisms responsible and amendable to therapy represents a growing challenge in medicine. At a cellular level, increased activity of the hexosamine biosynthesis pathway (HBP), a sensor of excess energy status, has been suggested to promote the exacerbation of insulin resistance through increasing adipose tissue and skeletal muscle membrane cholesterol content. This in turn compromises cortical filamentous actin structure necessary for proper incorporation of the insulin-sensitive glucose transporter GLUT4 into the plasma membrane. The current studies attempted to elucidate the mechanism by which hexosamines provoke membrane cholesterol toxicity and insulin resistance. In 3T3-L1 adipocytes cultured with pathophysiologic hyperinsulinemia to induce insulin resistance, increased HBP flux was observed. This occurred concomitant with gains in the mRNA and protein levels of HMG-CoA reductase (HMGR), the rate limiting enzyme in cholesterol synthesis. Mechanistically, immunoprecipitation demonstrated increased HBP-induced N-acetylglucosamine (O-GlcNAc) modification of specificity protein 1 (Sp1), a regulator of HMGR synthesis. This was associated with increased affinity toward and activity of Hmgcr, the gene encoding HMGR. Global HBP inhibition or Sp1 binding to DNA prevented membrane cholesterol accrual, filamentous actin loss, and glucose transport dysfunction. Furthermore, hyperinsulinemia and HBP activation impaired cholesterol efflux in adipocytes, exacerbating cholesterol toxicity and potentially contributing to cardiovascular disease. In this regard, chromium picolinate (CrPic), known to have beneficial effects on glucose and lipoprotein metabolism, improved cholesterol efflux and restored membrane cholesterol content. To test the role of membrane cholesterol accumulation in vivo, studies were conducted on C57Bl/6J mice fed a low or high fat diet. High fat feeding promoted increased HBP activity, membrane cholesterol accumulation, and insulin resistance. Supplementation of mice with CrPic in their drinking water (8µg/kg/day) countered these derangements and improved insulin sensitivity. Together, these data provide mechanistic insight for the role of membrane cholesterol stress in the development of insulin resistance, as well as cardiovascular disease, and highlight a novel therapeutic action of chromium entailing inhibition of the HBP pathway.Item The RGS-RhoGEFs control the amplitude of YAP1 activation by serum(Springer Nature, 2021-01-27) Lane, Brandon S.; Heller, Brigitte; Hollenberg, Morley D.; Wells, Clark D.; Biochemistry and Molecular Biology, School of MedicineActin-dependent mechanisms drive the nuclear translocation of Yap1 to enable its co-activation of transcription factors that induce pro-growth and survival programs. While Rho GTPases are necessary for the nuclear import of YAP1, the relevant Guanine Exchange Factors (GEFs) and GTPase Activating Proteins (GAPs) that connect this process to upstream signaling are not well defined. To this end, we measured the impact of expressing sixty-seven RhoGEFs and RhoGAPs on the YAP1 dependent activity of a TEAD element transcriptional reporter. Robust effects by all three members of the regulator of G-protein signaling (RGS) domain containing RhoGEFs (ArhGEF1, ArhGEF11 and ArhGEF12) prompted studies relating their known roles in serum signaling onto the regulation of Yap1. Under all conditions examined, ArhGEF12 preferentially mediated the activation of YAP1/TEAD by serum versus ArhGEF1 or ArhGEF11. Conversely, ArhGEF1 in multiple contexts inhibited both basal and serum elevated YAP1 activity through its GAP activity for Gα13. The sensitivity of such inhibition to cellular density and to low states of serum signaling supports that ArhGEF1 is a context dependent regulator of YAP1. Taken together, the relative activities of the RGS-RhoGEFs were found to dictate the degree to which serum signaling promotes YAP1 activity.Item The role of ADF and cofilin in auditory sensory cell development(2020-12) McGrath, Jamis; Perrin, Benjamin; Cummins, Theodore; Belecky-Adams, Teri; Balakrishnan, Lata; Zhao, BoOur ability to hear relies on sensory cells found in the inner ear that transduce sound into biological signals. Microvilli-like protrusions called stereocilia are bundled on the apical surfaces of these cells and allow them to respond to sound-evoked vibrations. The architecture of the stereocilia bundle is highly patterned to ensure normal hearing. Filaments of polymerized actin proteins are bundled in parallel into large cylindrical structures that define the dimensions of stereocilia. This network is then anchored to the cell by inserting into another actin-based structure called the cuticular plate, which forms a gel-like structure and facilitates the mechanical properties of the bundle. The shape of the bundle is determined through tissue-level and intrinsic polarization signaling pathways. Auditory brainstem-evoked response testing, immunofluorescence imaging, scanning electron microscopy, and biochemical labeling techniques were used to study how the ADF/cofilin family of actin filament severing and depolymerizing proteins contributes to the development of the stereocilia bundle. Loss of these proteins disrupts the normal bundle patterning process, changes the lengths and widths of stereocilia, and alters the regulation of filament ends near the ion channel at stereocilia tips that is responsible for mechanotransduction. The activity of this channel regulates ADF/cofilins and the actin at stereocilia tips. Aberrant actin growth in actin networks beneath the stereocilia bundle influences the bundle patterning process, causes dysmorphic bundles to form. This work identifies that ADF/cofilins are necessary during auditory sensory cell development to facilitate normal bundle patterning and establishes this protein family as a molecular link between mechanotransduction and stereocilia bundle maturation.