TY - JOUR
T1 - Myosin-X silencing in the trabecular meshwork suggests a role for tunneling nanotubes in outflow regulation
AU - Sun, Ying Ying
AU - Yang, Yong Feng
AU - Keller, Kate E.
N1 - Funding Information:
The authors thank Lions VisionGift (Portland, OR, USA) for facilitating the procurement of human cadaver eyes. Supported by NIH/NEI grants EY019643 (KEK), EY010572 (P30 Casey Eye Institute Core facility grant), and an unrestricted grant to the Casey Eye Institute from Research to Prevent Blindness (New York, NY, USA).
Funding Information:
The authors thank Lions VisionGift (Portland, OR, USA) for facilitating the procurement of human cadaver eyes. Supported by NIH/NEI grants EY019643 (KEK), EY010572 (P30 Casey Eye Institute Core facility grant), and an unrestricted grant to the Casey Eye Institute from Research to Prevent Blindness (New York, NY, USA). Disclosure: Y.Y. Sun, None; Y.-F. Yang, None; K.E. Keller, None
Publisher Copyright:
© 2019 The Authors.
PY - 2019/2
Y1 - 2019/2
N2 - PURPOSE. The actin cytoskeleton plays a key role in outflow regulation through the trabecular meshwork (TM). Although actin stress fibers are a target of glaucoma therapies, the role of other actin cellular structures is unclear. Myosin-X (Myo10) is an actin-binding protein that is involved in tunneling nanotube (TNT) and filopodia formation. Here, we inhibited Myo10 pharmacologically or by gene silencing to investigate the role of filopodia/TNTs in the TM. METHODS. Short hairpin RNA interference (RNAi) silencing lentivirus targeting myosin-X (shMyo10) was generated. Human anterior segments were perfused with shMyo10 or CK-666, an Arp2/3 inhibitor. Confocal microscopy investigated the colocalization of Myo10 with matrix metalloproteinase (MMPs). Western immunoblotting investigated the protein levels of MMPs and extracellular matrix (ECM) proteins. MMP activity and phagocytosis assays were performed. RESULTS. CK-666 and shMyo10-silencing lentivirus caused a significant reduction in outflow rates in anterior segment perfusion culture, an ex vivo method to study intraocular pressure regulation. In human TM cells, Myo10 colocalized with MMP2, MMP14, and cortactin in podosome-like structures, which function as regions of focal ECM degradation. Furthermore, MMP activity, thrombospondin-1 and SPARC protein levels were significantly reduced in the media of CK-666-treated and shMyo10-silenced TM cells. However, neither Myo10 silencing or CK-666 treatment significantly affected phagocytic uptake. CONCLUSIONS. Inhibiting filopodia/TNTs caused opposite effects on outflow compared with inhibiting stress fibers. Moreover, Myo10 may also play a role in focal ECM degradation in TM cells. Our results provide additional insight into the function of actin supramolecular assemblies and actin-binding proteins in outflow regulation.
AB - PURPOSE. The actin cytoskeleton plays a key role in outflow regulation through the trabecular meshwork (TM). Although actin stress fibers are a target of glaucoma therapies, the role of other actin cellular structures is unclear. Myosin-X (Myo10) is an actin-binding protein that is involved in tunneling nanotube (TNT) and filopodia formation. Here, we inhibited Myo10 pharmacologically or by gene silencing to investigate the role of filopodia/TNTs in the TM. METHODS. Short hairpin RNA interference (RNAi) silencing lentivirus targeting myosin-X (shMyo10) was generated. Human anterior segments were perfused with shMyo10 or CK-666, an Arp2/3 inhibitor. Confocal microscopy investigated the colocalization of Myo10 with matrix metalloproteinase (MMPs). Western immunoblotting investigated the protein levels of MMPs and extracellular matrix (ECM) proteins. MMP activity and phagocytosis assays were performed. RESULTS. CK-666 and shMyo10-silencing lentivirus caused a significant reduction in outflow rates in anterior segment perfusion culture, an ex vivo method to study intraocular pressure regulation. In human TM cells, Myo10 colocalized with MMP2, MMP14, and cortactin in podosome-like structures, which function as regions of focal ECM degradation. Furthermore, MMP activity, thrombospondin-1 and SPARC protein levels were significantly reduced in the media of CK-666-treated and shMyo10-silenced TM cells. However, neither Myo10 silencing or CK-666 treatment significantly affected phagocytic uptake. CONCLUSIONS. Inhibiting filopodia/TNTs caused opposite effects on outflow compared with inhibiting stress fibers. Moreover, Myo10 may also play a role in focal ECM degradation in TM cells. Our results provide additional insight into the function of actin supramolecular assemblies and actin-binding proteins in outflow regulation.
KW - Actin cytoskeleton
KW - Aqueous humor dynamics
KW - Extracellular matrix
KW - Trabecular meshwork
UR - http://www.scopus.com/inward/record.url?scp=85063390074&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85063390074&partnerID=8YFLogxK
U2 - 10.1167/iovs.18-26055
DO - 10.1167/iovs.18-26055
M3 - Article
C2 - 30807639
AN - SCOPUS:85063390074
SN - 0146-0404
VL - 60
SP - 843
EP - 851
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 2
ER -