Local Ras activation, PTEN pattern, and global actin flow in the chemotactic responses of oversized cells

Abstract

Chemotactic responses of eukaryotic cells require a signal processing system that translates an external gradient of attractant into directed motion. To challenge the response system to its limits, we increased the size of Dictyostelium discoideum cells by using electric-pulse-induced fusion. Large cells formed multiple protrusions at different sites along the gradient of chemoattractant, independently turned towards the gradient and competed with each other. Finally, these cells succeeded to re-establish polarity by coordinating front and tail activities. To analyse the responses, we combined two approaches, one aimed at local responses by visualising the dynamics of Ras activation at the front regions of reorientating cells, the other at global changes of polarity by monitoring front-to-tail-directed actin flow. Asymmetric Ras activation in turning protrusions underscores that gradients can be sensed locally and translated into orientation. Different to cells of normal size, the polarity of large cells is not linked to an increasing front-to-tail gradient of the PIP3-phosphatase PTEN. But even in large cells, the front communicates with the tail through an actin flow that might act as carrier of a protrusion inhibitor

Co-existence of Ras activation in a chemotactic signal transduction pathway and in an autonomous wave - forming system

“…For dual-color fluorescence imaging, cells of the AX2-214 strain of D. discoideum were transformed to express pair-wise mRFPM-LimED 31 and superfolding (sf) GFP-PHcrac, 32 mRFPM-LimED and GFP-Raf1-RBD, 1 the minimal Ras-binding domain (RBD) of human Raf proto-oncogene serine/threonine-protein kinase (Raf-1) as an activation-specific probe for Ras, 33,34 or mRFPM-Raf1-RBD and PTEN-sfGFP. 1 The double-transformants were cultivated in nutrient medium containing 10 mg/ml of blasticidin (Invitrogen, Life Technologies, Grand Island, NY, USA) and 10 mg/ml of G418 (Sigma-Aldrich, St. Louis, Missouri, USA) at 21 § 2 C.…”

“…In a recent paper we reported on the chemotactic responses of over-sized cells of Dictyostelium discoideum that we produced by electric-pulse induced fusion. 1 Here we wish to address one point that was only touched in the above paper: the different actions of two systems in a single cell that both involve the activation of Ras and the synthesis of PIP3. The first system is the signal processing network that enables cells to orientate in a gradient of chemoattractant.…”

Actin assembly states in Dictyostelium discoideum at different stages of development and during cellular stress

“…These primordia represent an activated state of the actin system that enables the cells to respond within seconds to local stimuli. The cell was produced by electric-pulse induced fusion of single cells (Lange et al, 2016), and expressed mRFP-LimED as a label for filamentous actin (red) and GFP-PHcrac as a marker for PIP3 (green). The confocal image (Zeiss LSM 780) shows that actin waves subdivide the membrane into distinct domains: in PIP3-rich inner territories circumscribed by a wave, and in the external area depleted of PIP3.…”

Rhythmicity and waves in the cortex of single cells

“…However, there are also exceptions. In Dictyostelium , the maximal velocity of the leading edge waves, which should also involve protrusion, can be as fast as 0.7 µm s −1 [ 86 , 87 ]. In addition, wave velocity could be faster when phosphatidylinositol-4,5-bisphosphate (PIP2) is depleted [ 88 ], which presumably reduces cortical actin.…”

“…A more direct parameter is perhaps the membrane curvature in the specific context of the membrane–cytoskeleton interface. Protrusive edges in Dictyostelium have a much less negative curvature compared with the lamellipodia in the fibroblast [ 86 ], which may explain why protrusive wave velocities are almost undiminished compared with those of the basal surface waves in Dictyostelium [ 87 ], but are much slower in other cell types.…”