Pvr and distinct downstream signaling factors are required for hemocyte spreading and epidermal wound closure at Drosophila larval wound sites.

Tissue injury is typically accompanied by inflammation. In Drosophila melanogaster larvae, wound-induced inflammation involves adhesive capture of hemocytes at the wound surface followed by hemocyte spreading to assume a flat, lamellar morphology. The factors that mediate this cell spreading at the wound site are not known.

larval epidermal cells only exhibit epidermal aging when they persist to the adult stage.

Holometabolous insects undergo a complete transformation of the body plan from the larval to the adult stage. In , this transformation includes replacement of larval epidermal cells (LECs) by adult epidermal cells (AECs). AECs in undergo a rapid and stereotyped aging program where they lose both cell membranes and nuclei.

Yorkie regulates epidermal wound healing in Drosophila larvae independently of cell proliferation and apoptosis.

Yorkie (Yki), the transcriptional co-activator of the Hippo signaling pathway, has well-characterized roles in balancing apoptosis and cell division during organ growth control. Yki is also required in diverse tissue regenerative contexts. In most cases this requirement reflects its well-characterized roles in balancing apoptosis and cell division.

Mitogen-activated Protein Kinase (MAPK) Activated by Prostaglandin E2 Phosphorylates Connexin 43 and Closes Osteocytic Hemichannels in Response to Continuous Flow Shear Stress.

Cx43 hemichannels serve as a portal for the release of prostaglandins, a critical process in mediating biological responses of mechanical loading on bone formation and remodeling. We have previously observed that fluid flow shear stress (FFSS) opens hemichannels; however, sustained FFSS results in hemichannel closure, as continuous opening of hemichannels is detrimental to cell viability and bone remodeling. However, the mechanism that regulates the closure of the hemichannels is unknown.

Connexin 43 channels are essential for normal bone structure and osteocyte viability.

Connexin (Cx) 43 serves important roles in bone function and development. Targeted deletion of Cx43 in osteoblasts or osteocytes leads to increased osteocyte apoptosis, osteoclast recruitment, and reduced biomechanical properties. Cx43 forms both gap junction channels and hemichannels, which mediate the communication between adjacent cells or between cell and extracellular environments, respectively.

Direct regulation of osteocytic connexin 43 hemichannels through AKT kinase activated by mechanical stimulation.

Connexin (Cx) 43 hemichannels in osteocytes are thought to play a critical role in releasing bone modulators in response to mechanical loading, a process important for bone formation and remodeling. However, the underlying mechanism that regulates the opening of mechanosensitive hemichannels is largely unknown. We have recently shown that Cx43 and integrin α5 interact directly with each other, and activation of PI3K appears to be required for Cx43 hemichannel opening by mechanical stimulation.

14-3-3θ facilitates plasma membrane delivery and function of mechanosensitive connexin 43 hemichannels.

Intracellular signaling in osteocytes activated by mechanical loading is important for bone formation and remodeling. These signaling events are mediated by small modulators released from Cx43 hemichannels (HC). We have recently shown that integrin α5 senses the mechanical stimulation and induces the opening of Cx43 HC; however, the underlying mechanism is unknown.

Using Drosophila larvae to study epidermal wound closure and inflammation.

This methods chapter describes two methods for creating epithelial wounds in Drosophila larvae: pinch and puncture wounding. It also covers protocols for visualizing epithelial wounds, either in a dissected whole mount preparation or, using transgenic reporter larvae, in a live whole mount preparation. Finally, useful transgenic lines for live genetic screening of genes required for wound closure or inflammation are described.

Mechanical stress-activated integrin α5β1 induces opening of connexin 43 hemichannels.

The connexin 43 (Cx43) hemichannel (HC) in the mechanosensory osteocytes is a major portal for the release of factors responsible for the anabolic effects of mechanical loading on bone formation and remodeling. However, little is known about how the Cx43 molecule responds to mechanical stimulation leading to the opening of the HC. Here, we demonstrate that integrin α5β1 interacts directly with Cx43 and that this interaction is required for mechanical stimulation-induced opening of the Cx43 HC.

Regulation of cellular function by connexin hemichannels.

Gap junctions and hemichannels are formed by a family of proteins called connexins. Till date up to twenty one different connexins have been characterized and their expression was observed to be spatio-temporally regulated. Gap junctions and hemichannels are involved in transfer of a variety of less than 1 kDa small molecules such as, ions, small metabolites, cAMP, ATP, IP3, prostaglandins, etc.

Dark horse in osteocyte biology: Glycocalyx around the dendrites is critical for osteocyte mechanosensing.

Osteocytes are considered as the major mechanosensory cells of the bone tissue that control the bone remodeling process. Since osteocytes are buried inside mineralized matrix, they maintain a strong communication network with other cells. Long dendritic processes of the osteocytes act as communication cables, conveying mechanical signals to the neighboring osteocytes and the cells on the bone surface; like osteoblasts and osteoclasts.

Dendritic processes of osteocytes are mechanotransducers that induce the opening of hemichannels.

Osteocytes with long dendritic processes are known to sense mechanical loading, which is essential for bone remodeling. There has been a long-standing debate with regard to which part(s) of osteocyte, the cell body versus the dendritic process, acts as a mechanical sensor. To address this question experimentally, we used a transwell filter system that differentiates the cell body from the dendritic processes.

Connexin 43 hemichannel opening associated with Prostaglandin E(2) release is adaptively regulated by mechanical stimulation.

Osteocytes present in the bone are known to be the major mechanosensory cells. Their involvement in mechanoregulation of bone remodeling is not yet clear. Osteocytes are connected with each other through gap junctions formed by Connexin 43 (Cx43).

Adaptation of connexin 43-hemichannel prostaglandin release to mechanical loading.

Bone tissues respond to mechanical loading/unloading regimens to accommodate (re)modeling requirements; however, the underlying molecular mechanism responsible for these responses is largely unknown. Previously, we reported that connexin (Cx) 43 hemichannels in mechanosensing osteocytes mediate the release of prostaglandin, PGE(2), a crucial factor for bone formation in response to anabolic loading. We show here that the opening of hemichannels and release of PGE(2) by shear stress were significantly inhibited by a potent antibody we developed that specifically blocks Cx43-hemichannels, but not gap junctions or other channels.

Roles of gap junctions and hemichannels in bone cell functions and in signal transmission of mechanical stress.

Gap junctions formed by connexins (Cx) play an important role in transmitting signals between bone cells such as osteoblasts and osteoclasts, cells responsible for bone formation and bone remodeling, respectively. Gap junction intercellular communication (GJIC) has been demonstrated to mediate the process of osteoblast differentiation and bone formation. Furthermore, GJIC propagates Ca2+ signaling, conveys anabolic effects of hormones and growth factors, and regulates gene transcription of osteoblast differentiation markers.