A dynamic partitioning mechanism polarizes membrane protein distribution

Abstract

The plasma membrane is widely regarded as the hub of the signal transduction network activities that drives numerous physiological responses, including cell polarity and migration. Yet, the symmetry breaking process in the membrane, that leads to dynamic compartmentalization of different proteins, remains poorly understood. Using multimodal live-cell imaging, here we first show that multiple endogenous and synthetic lipid-anchored proteins, despite maintaining stable tight association with the inner leaflet of the plasma membrane, were unexpectedly depleted from the membrane domains where the signaling network was spontaneously activated such as in the new protrusions as well as within the propagating ventral waves. Although their asymmetric patterns resembled those of standard peripheral "back" proteins such as PTEN, unlike the latter, these lipidated proteins did not dissociate from the membrane upon global receptor activation. Our experiments not only discounted the possibility of recurrent reversible translocation from membrane to cytosol as it occurs for weakly bound peripheral membrane proteins, but also ruled out the necessity of directed vesicular trafficking and cytoskeletal supramolecular structure-based restrictions in driving these dynamic symmetry breaking events. Selective photoconversion-based protein tracking assays suggested that these asymmetric patterns instead originate from the inherent ability of these membrane proteins to "dynamically partition" into distinct domains within the plane of the membrane. Consistently, single-molecule measurements showed that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane. When these profiles were incorporated into an excitable network-based stochastic reaction-diffusion model of the system, simulations revealed that our proposed "dynamic partitioning" mechanism is sufficient to give rise to familiar asymmetric propagating wave patterns. Moreover, we demonstrated that normally uniform integral and lipid-anchored membrane proteins inDictyosteliumand mammalian neutrophil cells can be induced to partition spatiotemporally to form polarized patterns, by optogenetically recruiting membrane domain-specific peptides to these proteins. Together, our results indicate dynamic partitioning as a new mechanism of plasma membrane organization, that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins in different physiological processes.

Complementary Cytoskeletal Feedback Loops Control Signal Transduction Excitability and Cell Polarity

“…Lower fluidity in regions with high cortical density may slow the diffusion of signaling activators, locally lowering excitability. Supporting this, multiple studies have reported lower membrane fluidity in back regions where actomyosin density should be high 61–63 . Another possibility is related to the finding that anionic lipids such as PI(4,5)P2 on the inner leaflet of the membrane inhibits Ras/PI3K signaling 6 .…”

Optogenetic modulation of guanine nucleotide exchange factors of Ras superfamily proteins directly controls cell shape and movement

“…Dictyostelium stable cell lines were generated by electroporation ( Li et al, 2021 ; Banerjee et al, 2022 ; Banerjee et al, 2023 ; Pal et al, 2023 ). Briefly, 1 × 10 7 cells were harvested, washed twice, and resuspended in 100 μL ice-cold H-50 buffer.…”

“…For imaging mammalian cells, both microscopes contained a temperature-controlled chamber held at 37°C and 5% CO 2 . Zeiss 800 or 780 was operated by ZEN Blue or Black software, respectively ( Kuhn et al, 2021 ; Banerjee et al, 2022 ; Banerjee et al, 2023 ; Pal et al, 2023 ).…”

“…For Figure 4B , cell was first segmented against the background with help of a custom code written in MATLAB 2019b ( https://www.mathworks.com/ ). Next, membrane kymograph was created from the segmented cell as described previously ( Banerjee et al, 2022 ; Banerjee et al, 2023 ; Pal et al, 2023 ). A linear color map for normalized intensities was used; blue denoted the lowest intensity whereas yellow denoted the highest.…”

Ras-mediated homeostatic control of front-back signaling dictates cell polarity

“…Medium along with unadhered cells was removed and 450 μL fresh supplemented SB was gently added to the well. Cells were allowed to recover for 1 hr before imaging 35,36 .…”