bioRxiv preprint doi: https://doi.org/10.1101/758896; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Phospholipase D Transduces Force to TREK-1 Channels in a Biological Membrane E. Nicholas Petersen1,4, Manasa Gudheti5, Mahmud Arif Pavel1, Keith R. Murphy2,3, William W. Ja2,3, Erik M. Jorgensen5, Scott B. Hansen1* 1Departments of Molecular Medicine, 2Department of Neuroscience, 3Center on Aging, 4Scripps Research Skaggs Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Scripps, Florida 33458, USA 5Department of Biology, Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112, USA *Correspondence: [email protected] ABSTRACT ordered proteins and lipids is a non-canonical sensor of mechanical force in biological The transduction of force into a biological signal is membranes4,5. Force was shown to disassemble critical to all living organisms. Recently, disruption and flatten caveolae4 and force disrupts ordered of ordered lipids has emerged as an ‘atypical’ force lipids and lipid clusters5. The ordered phase, sensor in biological membranes; however, sometimes called a lipid raft, is enriched in disruption has yet to link with canonical channel saturated lipids and palmitoylated proteins6,7 and is mechanosensation. Here we show that force- subject to disruption (see Supplemental Fig. 1a-b). induced disruption and lipid mixing activates TWIK- + In C2C12 muscle cells, chemical disruption related K channel (TREK-1), and that this releases a palmitoylated protein phospholipase D activation is dependent on phospholipase D2 (PLD2) from lipid rafts activating a mechano- (PLD2). PLD2 transduces the force into a chemical transduction pathway (Supplemental Fig. 1c-d).5 signal phosphatidic acid (PA) that is then sensed This mechanical activation of PLD2 is shown to by TREK-1 with a latency of <3 ms. TREK-1 then cause muscle cell differentiation5, produces a mechanically induced change in miacropinocytosis8, and may act as a force senor membrane potential. Hence, in a biological upstream of mTOR9. These data point to lipid membrane, we show the ordered lipid is the force domains as bona fide sensors of force. sensor, PLD2 is a chemical transducer, and the ‘mechanosensitive’ ion channel TREK-1 is a The downstream effector molecules of this downstream effector of mechanical transduction. non-canonical pathway have yet to be described Confirming this central role for PA singling in force for excitable systems. Here we hypothesize transduction, genetic deletion of PLD decreases anionic signaling lipids are a transducer of mechanosensitivity and pain thresholds in D. mechanical force to ion channels. Many ion melanogaster. channels and transporters are regulated by lipids and could serve as effector molecules of lipid raft Keywords: ion channel; lipids; phospholipase D; disruption. Additionally, in eukaryotic membrane; mechanosensation; kinetic; lipid rafts; membranes10,11 excess lipids and cellular INTRODUCTION cytoskeletal structures can resist tension on and propagation through plasma membranes12–15, All cells must sense force suggesting the non-canonical disruption (mechanosensation), not just sensory pain nerves, mechanism likely plays a prominent role for at including those that respond to the heartbeat, least some ion channel activation. Linking atypical skeletal muscle stretch, and epithelial cell raft mechanosensation directly to a canonical adhesion1–3, Recent studies show that disruption of bioRxiv preprint doi: https://doi.org/10.1101/758896; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. mechanosensitive channel would constitute a and force would be an endogenous disruptor of distinct mechanism separate from the traditional lipid order for most cell types. “force from lipid” model typical of sensory systems where the channel directly senses stretch16,17. To directly image force-induced disruption of lipid domains, we developed a process to chemically fix disrupted domains in the membrane + of differentiated C2C12 muscle cells (mouse TWIK-related K channel type 1 (TREK-1) myocytes) using a pump and shear chambers (ibidi is a mechanosensitive member of the two-pore- µ-Slide I0.4 parallel-plate) (Supplemental Fig. 2a). domain potassium (K2P) family that protects 18 We fixed the cells with a combination of both against pain , and it is one of many channels paraformaldehyde and glutaraldehyde and regulated by lipids suggesting it could be down confirmed by fluorescence recovery after stream of raft disruption. Here we show membrane photobleaching (FRAP) that are conditions inhibit disruption releases PLD2 and generates the extra-domain diffusion of GM1 lipids anionic lipid-signal phosphatidic acid (PA) that (Supplemental Fig. 2b). We tested muscle cells directly activates TREK-1. Blocking the synthesis since they are known to respond to force and our of PA completely blocks stretch activation of previous experiments suggested force disrupts TREK-1 in whole-cell patch-clamp their GM1 domains5. Furthermore muscle cells electrophysiology. We conclude that in biological endogenously express PLD and TREK-1 membranes, raft disruption and lipid mixing is a channels5,31 allowing for observation of their dominant mechanosensor for TREK-1 channels dynamics without overexpression artifacts. and the anionic lipid PA is an essential intermediary. First, we initiated domain disruption by pumping phosphate buffered saline (PBS) across the C2C12 cells with a calibrated shear force of 3 2 RESULTS dynes/cm , a physiologically relevant force to muscle cells5,31. Immediately after applying shear, Mechanical disruption of lipid rafts in muscles we infused fixative agents to the shear buffer which cells. allowed the cells to be fixed in a mechanically- stimulated state. Non-sheared control cells were The best studied raft domains contain grown and treated similarly on static coverslips saturated lipids, cholesterol, sphingomyelin, and without shear. Fixed cells, with and without shear, monosialotetrahexosylganglioside1 (GM1)7. Cells were then prepared for dSTORM imaging using contain a second class of lateral heterogeneity antibodies or cholera toxin B (CtxB) with high comprised of anionic lipids PIP and PIP , which 2 3 affinity corresponding to either PIP or GM1 cluster19–21 separate from GM1 domains and 2 domains respectively5. The amount of fluorescent separate from each other5,22,23 (Supplemental Fig. labeling remained relatively constant between S1a). Lipid domains are thought to be sub-100 nm shear and static cells (Supplemental Fig. 2c). diameter structures, may include protein interactions24, but even with super-resolution We found shear forces robustly disrupted imaging techniques such as direct stochastic both GM1 and PIP2 domain size in fixed C2C12 optical reconstruction microscopy (dSTORM) and cells (Fig. 1a-b). Interestingly, we observed that the stimulated emission depletion (STED)25–28 their shear specific disruption reduced GM1 domain 29,30 exact structure remains controversial . We size more than PIP2 domains. Prior to shear, the previously used dSTORM to image live- estimated size of GM1 domains were larger than spontaneous and chemical disruption of GM1 PIP2 domains (167±3 vs. 154±1 nm in diameter). 5 domains in muscle cells leading us to asked if After shear, GM1 domains were smaller than PIP2 force induced disruption could directly be domains (131±3 vs. 139±1 nm in diameter, Fig. 1c- measured with imaging. Imaging direct disruption d). Domain size was determined using image- of lipid domains by force is important since GM1 based clustering with identical threshold and PIP2 lipids are part of all cellular membranes bioRxiv preprint doi: https://doi.org/10.1101/758896; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. produce PA. In addition to binding PIP2, PLD2 also binds directly to the C- terminus of TREK-1 and activates the channel through local production of phosphatidic acid35 presumably in the disordered region of the cell membrane5. Previous work also implicated TREK-1 C- terminus in mechanosensitivity36. We reasoned mechanical PLD2 activation could transduce a component of force to TREK-1 in a biological Figure 1. Mechanical induced disruption of GM1 and PIP2 domains. (a-b) Representative images showing the effect of shear on the size of GM1 and PIP2 - membrane independent of labeled domains in muscle C2C12 cells. When shear force is applied the apparent size direct TREK-1 stretch. If of both domains decreases significantly (c) (** is p<0.01, **** is p<0.0001, n>800 rafts PLD2 and PA are from 5-6 cells). The dominant domain by area shifts from CTxB-labeled domains to PIP2 intermediaries in TREK-1 domains after shear is applied (d). activation, this mechanism would link atypical raft parameters for all images (see methods). The mechanosensation directly to a canonical absolute size has potential artifacts from the 32 mechanosensitive channel and support our analysis , however, the absolute size of rafts is proposed mixing and lipid-signaling mechanism for inconsequential to our hypothesis. Rather, the stretch activation. We