Kinetic disruption of lipid rafts is a mechanosensor for phospholipase D

Abstract

The sensing of physical force, mechanosensation, underlies two of five human senses-touch and hearing. How transduction of force in a membrane occurs remains unclear. We asked if a biological membrane could employ kinetic energy to transduce a signal absent tension. Here we show that lipid rafts are dynamic compartments that inactivate the signalling enzyme phospholipase D2 (PLD2) by sequestering the enzyme from its substrate. Mechanical disruption of the lipid rafts activates PLD2 by mixing the enzyme with its substrate to produce the signalling lipid phosphatidic acid (PA). We calculate a latency time of <650 μs for PLD activation by mixing. Our results establish a fast, non-tension mechanism for mechanotransduction where disruption of ordered lipids initiates a mechanosensitive signal for cell growth through mechanical mixing.

Figure 1. Live cell imaging of lipid raft disruption in C2C12 cells.

(a) Diagram of the two major effects resulting from forces applied to a membrane. (b) Diagram of kinetic hypothesis for mechanical activation. An enzyme localized to a lipid raft is sequestered away from its substrate. Mechanically induced translocation of the enzyme from the raft leads to substrate access and enzyme activation. (c–e) dSTORM imaging of live C2C12 cells. (c) Single frames showing assembly and disassembly of a ∼125 nm CTx-raft (cropped from Supplementary movie S1). (d) Time averaged CTx-raft localization (movie S1); rafts dynamics are outlined with hubs and highways observed during live imaging (30 s), scale bar is 3 μm. The hubs are areas of high probability for large raft assembly and disassembly, while the highways allow for transient ordered trafficking of small particles between hubs (white tracing). (e) Time-dependent localization maps showing ordered domains were localized before (left), but rarely after mild mβCD treatment (100 μM) (right). Colours represent time; t=0 (dark red) to t=2.5 min (white); scale bar is 3 μm.

Figure 2. Quantitative effect of membrane disruption on raft diameter.

(a) Lipid raft sizes were determined for CTxB and PIP₂ domains before and after treatment with mβCD (reported as means±s.d.) ***P<0.001, ****P<0.0001 by two-tailed Student's t-test. A reduction of cholesterol by mβCD shows a decrease in the overall size of CTx-rafts and an increase in the average diameter of PIP₂ domains. (b–d) Histograms of particle size distribution. (b) Analysis of PIP₂ domains after mβCD treatment shows the size increase occurs as a result of a shift in small, well-defined, particles (<100 nm) to a slightly larger diameter. (c) In contrast, CTx-rafts show little change in particles <100 nm in diameter and an almost total loss of a heterogeneous population of rafts >100 nm in diameter (see c. zoom). (d) Disruption of PLD2 labelled rafts was similar to CTx-rafts with the change occurring mostly as a result of the elimination of rafts >100 nm (see d. zoom).

Figure 3. Raft disruption translocates PLD from CTx-rafts to PIP₂ domains.

(a) Fluorescently labelled PLD2 enzyme (green) colocalizes (merged control) with CTxB-labelled cholesterol rafts (red) before, but not after, treatment with mβCD confirmed by cross-correlation analysis in b. In contrast, prior to mβCD treatment PLD2 localizes away from PIP₂ domains (cyan) (c), increasing in correlation only after treatment with mβCD (d). Bars in a and c represent 2 μm. (e) On the distal ends of cells, PIP₂ domains and PLD were observed in striped, concentrated regions. An overlay of high-density regions of PIP₂ and PLD2 domains highlights this observation. Bars in e represent 4 μm.

Figure 4. Mechanical disruption of lipid rafts activates PLD enzyme.

(a) shows the effect of shear force on PLD activity in C2C12 cells. (c) shows an increase in PLD activity due to mβCD alone. (b) and (d) are a quantification of (a) and (c), (n=4–8, **P<0.01, ***P<0.001, Student's t-test). (e) PLD2 mediates the transduction of force to trigger cell differentiation. Mechanical force or mβCD treatment alone is able to increase the rate of differentiation, whereas the PLD2 antagonist FIPI inhibits this differentiation (n=4–5). Error bars are reported as mean±s.e.m.

Figure 5. A kinetic (non-tension) mechanism for mechanosensation.

CTxB- and PIP₂ domains exist in tandem, often in localized regions at the distal ends of cells (a). The proximal separation of PLD and PIP₂ allows for a ‘primed state', decreasing the latency between PLD2 translocation from cholesterol rafts to enzymatic activation by PIP₂ (∼650 μs). (b) Mechanical force increases the kinetic energy in the membrane. The increase in kinetic energy overcomes the miscibility of the raft components, leading to raft disruption. During this disruption, PLD2 experiences increased access to PC and PIP₂ resulting in the increased synthesis of the signalling lipid, PA.