[Paper Review] Self Organized Criticality in the Neural Networks

M. N. Najafi|arXiv (Cornell University)|Jun 3, 2013

Neural Networks and Applications1 references3 citations

TL;DR

This study identifies RhoD as a key regulator of cytoneme-like protrusions in mammalian cells, activated by FGF2/4/8 signaling to induce actin polymerization via mDia3C, forming stable, motile protrusions that transport endosome-like vesicles and mediate intercellular communication. The RhoD-mDia3C axis is essential for FGF-induced protrusion formation and function.

ABSTRACT

The small GTPase RhoD regulates actin cytoskeleton to collapse actin stress fibers and focal adhesions, resulting in suppression of cell migration and cytokinesis. It also induces alignment of early endosomes along actin filaments and reduces their motility. We show here that a constitutively activated RhoD generated two types of actin-containing thin peripheral cellular protrusions distinct from Cdc42-induced filopodia. One was longer, almost straight, immotile, and sensitive to fixation, whereas the other was shorter, undulating, motile, and resistant to fixation. Moreover, cells expressing wild-type RhoD extended protrusions toward fibroblast growth factor (FGF) 2/4/8-coated beads. Stimulation of wild-type RhoD-expressing cells with these FGFs also caused formation of cellular protrusions. Nodules moved through the RhoD-induced longer protrusions, mainly toward the cell body. Exogenously expressed FGF receptor was associated with these moving nodules containing endosome-like vesicles. These results suggest that the protrusions are responsible for intercellular communication mediated by FGF and its receptor. Accordingly, the protrusions are morphologically and functionally equivalent to cytonemes. RhoD was activated by FGF2/4/8. Knockdown of RhoD interfered with FGF-induced protrusion formation. Activated RhoD specifically bound to mDia3C and facilitated actin polymerization together with mDia3C. mDia3C was localized to the tips or stems of the protrusions. In addition, constitutively activated mDia3C formed protrusions without RhoD or FGF stimulation. Knockdown of mDia3 obstructed RhoD-induced protrusion formation. These results imply that RhoD activated by FGF signaling forms cytoneme-like protrusions through activation of mDia3C, which induces actin filament formation.

Motivation & Objective

• To investigate the role of RhoD in regulating actin cytoskeleton dynamics during cell migration and cytokinesis.
• To determine how RhoD influences the formation and function of specialized actin-based protrusions in response to FGF signaling.
• To elucidate the molecular mechanism by which RhoD controls protrusion formation through interaction with mDia3C.

Proposed method

• Expression of constitutively active RhoD to induce distinct types of actin-containing protrusions in mammalian cells.
• Use of FGF-coated beads to stimulate wild-type RhoD-expressing cells and assess protrusion induction.
• Live-cell imaging and fixation experiments to characterize protrusion morphology, motility, and stability.
• Co-immunoprecipitation and pull-down assays to test RhoD binding to mDia3C.
• RNAi-mediated knockdown of RhoD or mDia3 to assess functional dependence of protrusion formation.
• Immunofluorescence and confocal microscopy to localize mDia3C to protrusion tips and stems.

Experimental results

Research questions

• RQ1 How does RhoD regulate the formation of actin-based protrusions distinct from filopodia?
• RQ2 What is the role of FGF2/4/8 in activating RhoD and inducing protrusion formation?
• RQ3 How does RhoD interact with mDia3C to promote actin polymerization in protrusions?
• RQ4 Are the RhoD-induced protrusions functionally equivalent to cytonemes in intercellular signaling?
• RQ5 What is the requirement of mDia3C in RhoD-mediated protrusion formation?

Key findings

• Constitutively active RhoD induced two morphologically distinct actin-containing protrusions: longer, straight, immotile, fixation-sensitive ones and shorter, undulating, motile, fixation-resistant ones.
• Wild-type RhoD-expressing cells extended protrusions toward FGF2/4/8-coated beads, and FGF stimulation alone induced protrusion formation.
• Nodules containing endosome-like vesicles moved along the longer RhoD-induced protrusions toward the cell body, suggesting transport function.
• Exogenously expressed FGF receptor was associated with these moving nodules, indicating functional intercellular signaling capacity.
• Activated RhoD specifically bound to mDia3C and promoted actin polymerization in cooperation with mDia3C.
• Knockdown of RhoD or mDia3 disrupted protrusion formation, and constitutively active mDia3C alone induced protrusions without RhoD or FGF input.