Optogenetic control of phosphoinositide metabolism

Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration.PLEKHG3 | cell polarity | F-actin binding | positive feedback | PI3K

Section: Plekhg3 Enhances Polarized Cell Migration Via a Positive Feementioning

confidence: 95%

PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front

Nguyen

Park

et al. 2016

“…Expression vectors encoding the inter-SH2 (iSH2) domain of regulatory subunit (p85α) fused with the PHR domain of Cry2 (Cry2-iSH2), and the PI(3,4,5)P 3 probe RFP-PH(Akt), and EGFP-PH(Akt) were kind gifts from Pietro DeCamilli's laboratory (Yale University, New Haven, CT) (47). CIBN-CAAX plasmid was obtained from Addgene.…”

Section: Methodsmentioning

confidence: 99%

“…S8C), indicating the sensitivity of cell spreading process to PI3K inhibition. Finally, to confirm that the spatially regulated membrane recruitment of Rac1 is a consequence of PI(3,4,5)P 3 polarization, we used an optogenetic strategy developed by Idevall-Hagren et al that allows for optical manipulation of local PI(3,4,5)P 3 synthesis by inducing recruitment of PI3K to the plasma membrane (47). In this system, cells coexpressing CIBN-GFP-CAAX and the Cry2PHR-iSH2(p85α) were illuminated with blue light, which causes translocation of Cry2PHR-iSH2(p85α) associated with the endogenous p110α to the membrane and induces synthesis of PI(3,4,5)P 3 .…”

Section: C-term Hypervariable Region Is Important For the Regulationmentioning

confidence: 99%

Single-molecule tracking of small GTPase Rac1 uncovers spatial regulation of membrane translocation and mechanism for polarized signaling

Das

Yin

Yang

et al. 2015

Polarized Rac1 signaling is a hallmark of many cellular functions, including cell adhesion, motility, and cell division. The two steps of Rac1 activation are its translocation to the plasma membrane and the exchange of nucleotide from GDP to GTP. It is, however, unclear whether these two processes are regulated independent of each other and what their respective roles are in polarization of Rac1 signaling. We designed a single-particle tracking (SPT) method to quantitatively analyze the kinetics of Rac1 membrane translocation in living cells. We found that the rate of Rac1 translocation was significantly elevated in protrusions during cell spreading on collagen. Furthermore, combining FRET sensor imaging with SPT measurements in the same cell, the recruitment of Rac1 was found to be polarized to an extent similar to that of the nucleotide exchange process. Statistical analysis of singlemolecule trajectories and optogenetic manipulation of membrane lipids revealed that Rac1 membrane translocation precedes nucleotide exchange, and is governed primarily by interactions with phospholipids, particularly PI(3,4,5)P 3 , instead of protein factors. Overall, the study highlights the significance of membrane translocation in spatial Rac1 signaling, which is in addition to the traditional view focusing primarily on GEF distribution and exchange reaction.super-resolution microscopy C ell polarization is critical for many biological processes, such as front−rear polarity during directed cell migration (chemotaxis, haptotaxis, wound healing, etc.), apical−basal polarity in epithelial cells, and axon specification in neuronal cells. The asymmetric distribution of signaling molecules, adhesion components, and cytoskeletal structures is important for the establishment of polarization. Among signaling proteins, the Rho family small GTPase Rac1 is ubiquitously required for cytoskeletal changes leading to a polarized morphology in many cells (1-4).Most small GTPases function as molecular switches, cycling between a GTP-bound active state and a GDP-bound inactive state. Activation of small GTPases typically requires guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP. Inactivation requires the inherent GTPase activity, enhanced by the GTPase Activating Proteins (GAPs) (5, 6). Thus, each Rac1 molecule continuously cycles between the active and inactive state during their intracellular existence.Besides the guanine nucleotide binding cycle, another cycle that governs Rac1 activity is the membrane/cytoplasm translocation cycle. Rac1 GTPase, as with many other small GTPases, is posttranslationally prenylated at its C terminus and is capable of binding to lipid bilayers (7,8). Under basal conditions, the majority of Rac1 localizes to the cytoplasm in complex with RhoGDI molecules, which block its interactions with GEFs and GAPs and its downstream effectors (9-12). To become active, Rac1 needs to translocate to the membrane and be free from RhoGDI binding, allowing for its activation by GEFs and its inter...

“…Chemical dimerizer and optogenetic approaches are options to manipulate lipid contents more rapidly, but they depend on cytosolic lipid-metabolizing enzymes. In the past, many applications therefore focused on phosphoinositides (3,4). A more general way to rapidly increase lipid concentration is the use of caged lipids.…”

mentioning

confidence: 99%

Trifunctional lipid probes for comprehensive studies of single lipid species in living cells

Höglinger

Nadler

Haberkant

et al. 2017

110

105

Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo-cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner.