Role of plasma membrane nano-structures during heat sensing

The study of the cellular stress response is of great importance to our understanding of how cells respond and adapt to various changes in their environment, especially during pathophysiological conditions. The classic heat shock (stress) response (HSR) was originally attributed to protein denaturation. However, induction of the heat shock protein (Hsp) molecular chaperones occurs in many circumstances where no protein denaturation is observed. Considerable evidence has been accumulated in favor of the "Membrane Sensor Hypothesis", which predicts that the level and ratio of Hsps can be modulated as a result of changes to the plasma membrane composition and/or organization. In order to understand the membrane associated stress sensing and signaling events it is of great importance to quantify how the membrane ultra-structure modulates signaling and how the signaling influences this ultra-structure. Our project will focus on the cell-to-cell variability in the heat shock response; in particular, we will monitor changes in protein and lipid levels as well as changes in the plasma membrane organization upon heat shock, and investigate how these are interrelated at the single cell level. To achieve this goal, we will combine the complementary expertise of our laboratories in the fields of molecular stress biology (Szeged) and single molecule microscopy (Vienna). Specifically, detailed lipidomic and proteomic analysis will be used in conjunction with single molecule microscopy techniques and image FCS. Our specific objectives are to (i) link the population heterogeneity of heat sensing with the membrane nanoscopic organization and dynamics, to (ii) elucidate the correlation between membrane composition and structural responsiveness, to (iii) identify the sequence of early events during temperature stress, to (iv) identify novel membrane localized heat sensors and to (v) investigate the role of the cell cycle phase in the cells ability to sense heat stress.

How does a biological cell sense temperature? In the original concept of a heat shock response, the sensor function of elevated temperatures was ascribed to the denaturation of certain molecules. However, denaturation of proteins does not occur at mild heat shock conditions, in which fever-like temperatures 2-4C above the physiological body temperature cause a reaction of the cell. Recently, considerable evidence has accumulated, that the plasma membrane of the cell itself could be the sensor. Small alterations to the plasma membrane have been shown to change the level of heat shock proteins, the latter being the first responders for counteracting temperature-induced changes in the cell. In favour of this membrane-sensor hypothesis, we were interested, whether and how nanometer-sized domains on the outside of the plasma membrane of living cells are involved in sensing the temperature. We applied a recently developed single molecule fluorescence microscopy technique which enabled the detection of these nano structures on the surface of living cells. Mobile nano structures were detected by their property to stably confine lipid-anchored fluorescent proteins. Adaptations of our approach enabled the direct observation of nano structure disintegration and reformation after temperature increase and decrease, respectively. Interestingly, the time course of both events was much longer than the time needed for changing the temperatures. Together with observed changes in endo- and exocytosis of nano structures upon temperature variation we concluded, that an indirect effect causes nano structure re-arrangement, rather than a direct effect of the temperature on the molecules involved. Our findings were also supported by the induction of increased Calcium levels in the cells interior, which could be the point of origin for observed plasma membrane changes. Next, we addressed the influence of oxidized phospholipids an important player in the development of atherosclerosis - if present in the cellular plasma membrane. Using our model system, we could show, that nano structures are disintegrated upon the presence of two different oxidized phospholipid species. Disintegration was dose- dependent and correlated with a death signal of the cell. Again, our studies could show, that disruption of nanostructures is not a direct effect by the sheer presence of the oxidized lipids and concomitant interaction with nanostructure constituents, but rather an indirect mechanism involving an enzyme located at the cells interior.