Fig 2: 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.

Fig 3: Quantitative effect of membrane disruption on raft diameter.(a) Lipid raft sizes were determined for CTxB and PIP2 domains before and after treatment with mßCD (reported as means±s.d.) ***P<0.001, ****P<0.0001 by two-tailed Student's t-test. A reduction of cholesterol by mßCD 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 mßCD 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).

Fig 4: Downregulation of hLKB1 correlates with increased mTOR signalling in malignant melanoma overexpressing PLD2.Immunostainings of paraffin-embedded tissue sections of normal skin (a–d), neavi (a'–d') and primary malignant melanoma tumours (a''–d''). (a) hLKB1 is highly expressed in melanocytes in the stratum basale of the epidermis in situ (a, arrows), and still detectable at substantial levels (a') whereas it is almost undetectable in the majority of malignant melanoma (a''). (b) PLD2 is not detectable in melanocytes of healthy skin sections (b, arrows) but becomes overexpressed in naevi (b') and primary tumours (b''). (c) Akt is activated (indicated by staining of phospho Akt, pAkt) in naevi (c') and malignant melanoma (c''), whereas it is not detectable in melanocytes of healthy skin samples (c). (d) Melanocytes in healthy skin biopsies (d) show only faint nuclear staining for phospho S6K (pS6K), which was used as to evaluate mTOR activation. Naevi (d') exhibit only very few pS6K positive cells, whereas malignant melanoma show strong cytoplasmic and nuclear staining of pS6K (d''). (e) Quantification of melanoma biopsies scored for the indicated correlations. ‘Others’ are tumours, which do not exhibit increased phospho-Akt or PLD2 staining.

Fig 5: 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 mßCD 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 mßCD 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.