Rac1 activation can generate untemplated, lamellar membrane ruffles

F Leyden,U K Moorthi,U K Moorthi,H M York,H M York,E P Petrov,S Uthishtran,S Arumugam,S Arumugam,S Arumugam,S Arumugam,T Bornschlögl,H Gandhi,H Gandhi,A Patil,A Patil

doi:10.1186/s12915-021-00997-3

Abstract

BackgroundMembrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. Small GTPase Rac1 controls the membrane protrusions that form and encapsulate extracellular volumes to perform pinocytic or phagocytic functions.ResultsHere, capitalizing on rapid volumetric imaging capabilities of lattice light-sheet microscopy (LLSM), we describe optogenetic approaches using photoactivable Rac1 (PA-Rac1) for controlled ruffle generation. We demonstrate that PA-Rac1 activation needs to be continuous, suggesting a threshold local concentration for sustained actin polymerization leading to ruffling. We show that Rac1 activation leads to actin assembly at the dorsal surface of the cell membrane that result in sheet-like protrusion formation without any requirement of a template. Further, this approach can be used to study the complex morpho-dynamics of the protrusions or to investigate specific proteins that may be enriched in the ruffles. Deactivating PA-Rac1 leads to complex contractile processes resulting in formation of macropinosomes. Using multicolour imaging in combination with these approaches, we find that Myo1e specifically is enriched in the ruffles.ConclusionsCombining LLSM and optogenetics enables superior spatial and temporal control for studying such dynamic mechanisms. Demonstrated here, the techniques implemented provide insight into the complex nature of the molecular interplay involved in dynamic actin machinery, revealing that Rac1 activation can generate untemplated, lamellar protrusions.

Highlights

Membrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell
Continuous activation of Rac1 activity is required for effective ruffle formation To visualize dorsal membrane ruffling on-demand, we imaged human retinal pigmented epithelial (RPE1) cells transiently transfected with pTriExmCherry photoactivable Rac1 (PA-Rac1) as previously described [15, 16]
We attempted to activate PA-Rac1 using a 445-nm laser passing through the spatial light modulator and excitation objective to produce a ‘full length’ or ‘quarter length’ multi-bessel beam excitation profile (Fig. 1b, c)

Summary

Introduction

Membrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. A variety of molecular modules, The formation of dorsal ruffles or any elaborate membrane protrusion structure on the dorsal surface of the cell is a sophisticated multifaceted event that involves complex spatio-temporal activity of protein machineries [7]. It is poorly understood how the ‘lamella-machinery’ utilizes biochemical and biophysical interplay to execute. PA-Rac expressed in Raw264 macrophages induced membrane ruffling and macropinocytic cup formation [16] They further demonstrate that deactivation of Rac is crucial for the progress of pinocytic cup closure. Fritz-Laylin et al used LLSM to image neutrophil-like HL-60 cells and found two important aspects of lamellar protrusions that formed in the absence of any substrate interactions: (1) free standing lamellae combine to give rise to complex geometries that are essentially lamellar and (2) lamellar protrusions do not require any ‘surface template’ [18]

Methods

Results

Conclusion