Microinjection of recombinant p21rho induces rapid changes in cell morphology.

Abstract

Abstract. The rho proteins, p21 ~, are ubiquitously expressed guanine nucleotide binding proteins with ,'o30% amino acid homology to p21'% but their biochemical function is unknown. We show here that microinjection of constitutively activated recombinant rho protein (Vall4rho) into subconfluent cells induces dramatic changes in cell morphology: 15-30 min after injection cells adopt a distinct and novel phenotype with a contracted cell body and finger-like processes still adherent to the substratum. Ribosylation of Vall4rho with the ADP-ribosyltransferase C3 from clostridium botulinum, before microinjection, renders the protein biologically inactive, but it has no effect on either its intrinsic biochemical properties or on its interaction with the GTPase activating protein, rho GAP. Microinjection of ribosylated normal rho, on the other hand, has a similar effect to injection of C3 transferase and induces complete rounding up of cells. We also report striking biochemical changes in actin filament organization when contact-inhibited quiescent 3T3 cells are injected with Vall4rho protein. The effects induced by activation or inactivation of p21 ~ described here, suggest that the biological function of this protein is to control some aspect of cytoskeletal organization.

A pharmacological activator of AMP-activated protein kinase (AMPK) induces astrocyte stellation

“…Merely inhibiting Rho and/or its downstream effectors is sufficient to induce stellation in astrocytes [1,44]. To directly test whether AICAR-induced stellation was upstream of RhoA, we transiently transfected cultured rat cortical astrocytes with a plasmid expressing constitutively active RhoA (RhoV14) [40]. In cells transfected with RhoV14, AICAR-induced stellation was completely prevented, in contrast to cells transfected with GFP plasmid as a control (Fig.…”

“…Plasmid DNA expressing myc-tagged constitutively active RhoA (RhoV14), a gift from Dr. Alan Hall (Memorial Sloan-Kettering Cancer Center), was created as described previously [40]. The pmaxGFP control plasmid was from Amaxa (Gaithersburg, MD, USA).…”

Activation of the non-canonical Dvl–Rac1–JNK pathway by Frizzled homologue 10 in human synovial sarcoma

“…Rho GTPase family proteins are known to be the key regulators of actin cytoskeleton dynamics and to play critical roles in planar cell polarity/non-canonical WNTFrizzled cascade (Paterson et al, 1990;Hall, 1998;Wiggan and Hamel, 2002 Upregulation of JNK phosphorylation was observed in primary synovial sarcomas as well as in synovial sarcoma cell lines. The Rac1-JNK cascade was considered to be activated in the presence of FZD10, but this activation was blocked by the knockdown of either Dvl3 or Dvl2 as well as by the knockdown of FZD10 (Figure 3).…”

α-Catulin, a Rho signalling component, can regulate NF-κB through binding to IKK-β, and confers resistance to apoptosis

“…Concerning the cellular function of a-catulin, the Rho family GTPases have been recognized as key regulators of actin cytoskeleton reorganization, formation of focal adhesions and several aspects of cell shape remodelling, such as the one occurring during cell migration (Paterson et al, 1990;Ridley and Hall, 1992). To assess whether a-catulin is involved in these processes, we performed a cell migration assay.…”