Cathepsin K Regulates Intraocular Pressure by Modulating Extracellular Matrix Remodeling and Actin-Bundling in the Trabecular Meshwork Outflow Pathway

The homeostasis of extracellular matrix (ECM) and actin dynamics in the trabecular meshwork (TM) outflow pathway plays a critical role in intraocular pressure (IOP) regulation. We studied the role of cathepsin K (CTSK), a lysosomal cysteine protease and a potent collagenase, on ECM modulation and actin cytoskeleton rearrangements in the TM outflow pathway and the regulation of IOP. Initially, we found that CTSK was negatively regulated by pathological stressors known to elevate IOP. Further, inactivating CTSK using balicatib, a pharmacological cell-permeable inhibitor of CTSK, resulted in IOP elevation due to increased levels and excessive deposition of ECM-like collagen-1A in the TM outflow pathway. The loss of CTSK activity resulted in actin-bundling via fascin and vinculin reorganization and by inhibiting actin depolymerization via phospho-cofilin. Contrarily, constitutive expression of CTSK decreased ECM and increased actin depolymerization by decreasing phospho-cofilin, negatively regulated the availability of active TGFβ2, and reduced the levels of alpha-smooth muscle actin (αSMA), indicating an antifibrotic action of CTSK. In conclusion, these observations, for the first time, demonstrate the significance of CTSK in IOP regulation by maintaining the ECM homeostasis and actin cytoskeleton-mediated contractile properties of the TM outflow pathway.

Treponema denticola-Induced RASA4 Upregulation Mediates Cytoskeletal Dysfunction and MMP-2 Activity in Periodontal Fibroblasts

The periodontal complex consists of the periodontal ligament (PDL), alveolar bone, and cementum, which work together to turn mechanical load into biological responses that are responsible for maintaining a homeostatic environment. However oral microbes, under conditions of dysbiosis, may challenge the actin dynamic properties of the PDL in the context of periodontal disease. To study this process, we examined host-microbial interactions in the context of the periodontium via molecular and functional cell assays and showed that human PDL cell interactions with Treponema denticola induce actin depolymerization through a novel actin reorganization signaling mechanism. This actin reorganization mechanism and loss of cell adhesion is a pathological response characterized by an initial upregulation of RASA4 mRNA expression resulting in an increase in matrix metalloproteinase-2 activity. This mechanism is specific to the T. denticola effector protein, dentilisin, thereby uncovering a novel effect for Treponema denticola-mediated RASA4 transcriptional activation and actin depolymerization in primary human PDL cells.

Arabidopsis ADF7 inhibits VLN1 to regulate actin filament dynamics and ROS accumulation in root hair development responses to osmotic stress

Actin dynamics are essential for root hair development, however, the underlying molecular mechanisms of actin binding protein cooperation and plant abiotic stress responses are largely unknown. Here, genetic analysis displayed that actin depolymerizing protein ADF7 and actin bundling protein VLN1 are positively and negatively involved in root hair development in Arabidopsis, respectively. Moreover, ADF7 acts upstream of VLN1 in root hair development by the analysis of RT-qPCR, Gus staining, Western blot and genetics. The observation of F-actin dynamics shows that ADF7 inhibits VLN1, leading to the decline of filament actin (F-actin) bundling and thick bundle formation and the increase of F-actin turnover and depolymerization in epidermal cells of root apices. Actin pharmacological experiments confirm that ADF7 and VLN1 are via regulating F-actin dynamics to active root hair development. Furthermore, F-actin depolymerization coregulated by ADF7 and VLN1 elevates the reactive oxygen species (ROS) level in root tips. Additionally, F-actin depolymerization and ROS accumulation coregulated by ADF7 and VLN1 are involved in osmotic stress-induced root hair development. Our work reveals that ADF7 inhibits VLN1 to induce F-actin turnover and depolymerization and ROS level in root tips, which play an important role in root hair formation responses to osmotic stress .

Rab14/MACF2 Complex Regulates Endosomal Targeting During Cytokinesis

Abscission is a complex cellular process that is required for mitotic division. It is well-established that coordinated and localized changes in actin and microtubule dynamics are vital for cytokinetic ring formation, as well as establishment of the abscission site. Actin cytoskeleton reorganization during abscission would not be possible without the interplay between Rab11- and Rab35-containing endosomes and their effector proteins, whose roles in regulating endocytic pathways at the cleavage furrow have now been studied extensively. Here, we identified Rab14 as a novel regulator of cytokinesis. We demonstrate that depletion of Rab14 causes either cytokinesis failure or significantly prolongs division time. We show that Rab14 contributes to the efficiency of recruiting Rab11-endosomes to the ICB microtubules and that Rab14 knockout leads to inhibition of actin clearance at the abscission site. Finally, we demonstrate that Rab14 binds to microtubule minus-end interacting MACF2/CAMSAP3 complex and that this binding affects targeting of endosomes to the ICB microtubules. Collectively, our data identified Rab14 and MACF2/CAMSAP3 as proteins that regulate actin depolymerization and endosome targeting during cytokinesis. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

The Cardioprotective PKA-Mediated Hsp20 Phosphorylation Modulates Protein Associations Regulating Cytoskeletal Dynamics

The cytoskeleton has a primary role in cardiomyocyte function, including the response to mechanical stimuli and injury. The small heat shock protein 20 (Hsp20) conveys protective effects in cardiac muscle that are linked to serine-16 (Ser16) Hsp20 phosphorylation by stress-induced PKA, but the link between Hsp20 and the cytoskeleton remains poorly understood. Herein, we demonstrate a physical and functional interaction of Hsp20 with the cytoskeletal protein 14-3-3. We show that, upon phosphorylation at Ser16, Hsp20 translocates from the cytosol to the cytoskeleton where it binds to 14-3-3. This leads to dissociation of 14-3-3 from the F-actin depolymerization regulator cofilin-2 (CFL2) and enhanced F-actin depolymerization. Importantly, we demonstrate that the P20L Hsp20 mutation associated with dilated cardiomyopathy exhibits reduced physical interaction with 14-3-3 due to diminished Ser16 phosphorylation, with subsequent failure to translocate to the cytoskeleton and inability to disassemble the 14-3-3/CFL2 complex. The topological sequestration of Hsp20 P20L ultimately results in impaired regulation of F-actin dynamics, an effect implicated in loss of cytoskeletal integrity and amelioration of the cardioprotective functions of Hsp20. These findings underscore the significance of Hsp20 phosphorylation in the regulation of actin cytoskeleton dynamics, with important implications in cardiac muscle physiology and pathophysiology.

The Role of the Integrated Stress Response and the Actin Cytoskeleton During Wound Healing

Background and Hypothesis: Chronic cutaneous wounds are a serious health concern afflicting millions of people. One of the primary factors preventing the closure of chronic wounds is the inability of keratinocytes to migrate across the wound bed. Epidermal keratinocytes migrate in a cohesive manner known as the keratinocyte collective cell migration (KCCM). Our lab has demonstrated that the integrated stress response (ISR) plays a key role in the KCCM. The ISR is initiated by stress-sensitive kinases, such as GCN2, and results in decreased global protein synthesis while preferentially increasing the translation of mRNAs encoding cytoprotective proteins. Wound repair also relies on the actin cytoskeleton, but the crosstalk between actin and the ISR is not well established. We hypothesize that the interaction between the ISR and the actin cytoskeleton is critical for KCCM during wound healing. Methods: Cutaneous wound healing was approximated in vitro using the KCCM-dependent scratch assay. Wild-type (WT) and GCN2-deleted (KO) keratinocytes were grown on coverslips, differentiated, scratched, and harvested at different times post-wounding. F-actin and vimentin (VIM) expression was monitored over time using fluorescent phalloidin-488 and immunofluorescence. In addition, WT keratinocytes were treated with actin-depolymerizing drugs and induction of ISR was measured using immunoblots. Results: Depolymerization of F-actin was observed along the leading edge of both wounded WT and GCN2-KO keratinocytes immediately following wounding. WT keratinocytes upregulated VIM expression at the leading edge whereas VIM expression remained unchanged in the wounded GCN2-KO keratinocytes. Treatment with latrunculin B and cytochalasin D, which both result in actin depolymerization, induced GCN2 phosphorylation in the differentiated WT keratinocytes. Conclusion and Potential Impact: F-actin depolymerization elicits a GCN2-mediated induction of the ISR. GCN2 and the ISR are critical components of the cutaneous wound repair process and their crosstalk with the actin cytoskeleton may serve as a novel therapeutic target in the treatment of chronic wounds.

The adaptor protein APPL2 controls glucose-stimulated insulin secretion via F-actin remodeling in pancreatic β-cells

Filamentous actin (F-actin) cytoskeletal remodeling is critical for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells, and its dysregulation causes type 2 diabetes. The adaptor protein APPL1 promotes first-phase GSIS by up-regulating solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein expression. However, whether APPL2 (a close homology of APPL1 with the same domain organization) plays a role in β-cell functions is unknown. Here, we show that APPL2 enhances GSIS by promoting F-actin remodeling via the small GTPase Rac1 in pancreatic β-cells. β-cell specific abrogation of APPL2 impaired GSIS, leading to glucose intolerance in mice. APPL2 deficiency largely abolished glucose-induced first- and second-phase insulin secretion in pancreatic islets. Real-time live-cell imaging and phalloidin staining revealed that APPL2 deficiency abolished glucose-induced F-actin depolymerization in pancreatic islets. Likewise, knockdown of APPL2 expression impaired glucose-stimulated F-actin depolymerization and subsequent insulin secretion in INS-1E cells, which were attributable to the impairment of Ras-related C3 botulinum toxin substrate 1 (Rac1) activation. Treatment with the F-actin depolymerization chemical compounds or overexpression of gelsolin (a F-actin remodeling protein) rescued APPL2 deficiency-induced defective GSIS. In addition, APPL2 interacted with Rac GTPase activating protein 1 (RacGAP1) in a glucose-dependent manner via the bin/amphiphysin/rvs-pleckstrin homology (BAR-PH) domain of APPL2 in INS-1E cells and HEK293 cells. Concomitant knockdown of RacGAP1 expression reverted APPL2 deficiency-induced defective GSIS, F-actin remodeling, and Rac1 activation in INS-1E cells. Our data indicate that APPL2 interacts with RacGAP1 and suppresses its negative action on Rac1 activity and F-actin depolymerization thereby enhancing GSIS in pancreatic β-cells.

Inflammation induced-PINCH expression leads to actin depolymerization and mitochondrial mislocalization in neurons

Diseases and disorders with a chronic neuroinflammatory component are often linked with changes in brain metabolism. Among neurodegenerative disorders, people living with human immunodeficiency virus (HIV) and Alzheimer’s disease (AD) are particularly vulnerable to metabolic disturbances, but mechanistic connections of inflammation, neurodegeneration and bioenergetic deficits in the central nervous system (CNS) are poorly defined. The particularly interesting new cystine histidine-rich protein called PINCH is nearly undetectable in healthy mature neurons, but is robustly expressed in tauopathy-associated neurodegenerative diseases including HIV infection and AD. Although robust PINCH expression has been reported in neurons in the brains of patients with HIV and AD, the molecular mechanisms and cellular consequences of increased PINCH expression in CNS disease was not known. In this context, we have identified the transcription factor responsible for PINCH induction in neuroinflammatory conditions and the effects of increased PINCH expression in neurons. Given that AD and neuroHIV share pathological features including cognitive impairment with chronic neuroinflammation, TNFa plays an important role in neurodegenerative processes. The viral protein Tat, is produced in the brain and is one of the main drivers of neuroinflammation and strongly induces TNFa. Our data show that TNFα-mediated activation of MEF2A via increased cellular calcium induces PINCH. In turn, this leads to disruption of the PINCH-ILK-Parvin ternary complex, cofilin activation by Tesk1 inactivation, and actin depolymerization. Disruption of actin led to perinuclear mislocalization of mitochondria by destabilizing the kinesin-dependent mitochondrial transport machinery resulting in impaired neuronal metabolism. Blocking TNFα-induced PINCH preserves mitochondrial localization and maintains metabolic functioning. These data report for the first time mechanistic and biological consequences of PINCH expression in neurons in the CNS in diseases with a chronic neuroinflammatory component. These findings point to maintenance of PINCH at normal physiological levels as a new therapeutic target for neurodegenerative diseases with impaired metabolism.