Control of T lymphocyte morphology by the GTPase Rho

Abstract

Background: Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton.

Results: Inactivation of the GTPase Rho in the human T lymphocytic cell line HPB-ALL does not inhibit constitutively high adhesion to the integrin beta1 substrate fibronectin. It did however result in the aberrant extension of finger-like dendritic processes on the substrates VCAM-1, Fn, and mAb specific to beta1 integrins. Time-lapse video microscopy demonstrated that C3 induced extensions were primarily the result of an altered pseudopod elongation rather than retraction. Once the stellate pseudopodia extended, none retracted, and cells became completely immobile. Filipodial structures were absent and the dendritic-like processes in C3 treated cells were rich in filamentous actin. Immunolocalization of RhoA in untreated HPB-ALL cells spreading on fibronectin demonstrated a diffuse staining pattern within the pseudopodia. In C3 treated cells, clusters of RhoA were pronounced and localized within the altered extensions.

Conclusions: GTPase Rho is actively involved in the regulation of T lymphocyte morphology and motility.

Figure 1

A. Western blot of RhoA protein in whole cell lysates of the T cell lines HPB-ALL and CEM. B. Electroporation of C3 efficiently ADP-ribosylates Rho in the T cell line HPB-ALL. Cells were untreated, electroporated in the presence of C3 (25 μg/ml), or electroporated in control buffer. After a 1 hr incubation at 37°C, cells were further ADP-ribosylated as described in Experimental Procedures by the addition of exogenous C3 and [³²P]-NAD after cell disruption.

Figure 2

HPB-ALL adhesion to the extracellular matrix component fibronectin. HPB-ALL cells were either electroporated with C3 (25 μg/ml) or mock electroporated and adhesion to Fn was compared. There was no decrease in the adhesion of C3 treated HPB-ALL cells. The standard deviation was less than 10%.

Figure 3

ADP-ribosylation of Rho alters HPB-ALL spreading on β1 integrin substrates. Cells were plated on BSA (panels 1–3), Fn (panels 4–6), VCAM-1 (panels 7–9), anti-β1 mAb 33B6 (panels 10–12), or poly-L-lysine (panels 13–15). HPB-ALL cells were electroporated with 25 μg/ml C3 (right column), 25 μg/ml BSA (middle column), or untreated (left column). Initial magnification was 200 ×. Experiments in panels 1–9 and panels 10–15 were performed separately. The images are representative of at least four separate experiments.

Figure 4

Punctate staining pattern of RhoA in untreated and C3 treated HPB-ALL cells. Cells were stained for RhoA as described in the Material and Methods. Untreated cells are in the left panels, and C3 treated cells are represented in the right panels. Arrows indicate punctate patterns of RhoA in HPB-ALL cells spreading on the extracellular matrix component fibronectin.

Figure 5

TRITC-phalloidin staining demonstrates that altered pseudopodia are enriched for filamentous actin. Cells were electroporated with control buffer (A), or electroporated with C3 (25 μg/ml, B, C) and plated on VCAM-1 that had been immobilized on glass coverslips.

Figure 6

Video time lapse microscopy demonstrates that the morphologically distinct HPB-ALL pseudopodia are due to aberrant extension, rather than aberrant pseudopodial retraction. Cells were "untreated," subjected to electroporation in control buffer ("mock electroporated"), or electroporated in the presence of C3 (25 μg/ml, "C3 electroporated"). Cells were then plated onto VCAM-1 and let spread for 1 hr (as described in Materials and Methods) while being recorded by a CCD camera and time lapse VCR. Numbers in lower left corners of images denote time elapsed in minutes. Arrowheads identify pseudopodia of spreading cells. Initial magnification was 400×.