(428i) Cellular Stress Disrupts Intracellular Transport Machinery

Motor-driven transport of endocytic vesicles plays a key role in intracellular trafficking of metabolic and therapeutic macromolecules. Transport of endocytic vesicles is one of the most fundamental processes in cellular metabolism and is tightly regulated. In this context, we questioned whether and how externally induced stresses perturb intracellular trafficking. This question is relevant to several pathological conditions such as cancer which impose stresses on cells or environmental conditions such as ischemia or mechanical damage that also impose stresses on cells.

We studied microtubule dependent active transport of endosomes as a model for intracellular transport. Adherent skin fibroblast cells were exposed to mechanical or thermal shock by a brief exposure to ultrasound (50 kHz, 0.7 W/cm2, 1 minute) or heat (45 C for 10 minutes) respectively. Video microscopy of cells with fluorescently labeled endosomes was performed just before and immediately after, as well as 30 minutes, 1 hour and 4 hours after the stress. Tracking of the endosomal population revealed a drastic and immediate slowing down of motor-driven transport after cellular shock which was slowly restored after 4 hours. Parameters characterizing active transport, such as temporal scaling of mean squared displacement, endosomal velocity and run-length distribution, were calculated and it was confirmed that stressed cells exhibit dominantly diffusive transport of endosomes compared to active transport in normal cells. For those endosomes in stressed cells that did show active transport, shorter and higher average number of runs per trajectory were observed compared to non-stressed cells.

To fundamentally understand the biological mechanisms behind the relationship between cellular stress and intracellular transport, we focused on examining the microtubule assembly and role of heat-shock-proteins in these cells. Both ultrasound and hyperthermia caused disorganization and reduction in microtubule network-density, which also followed a similar recovery profile. This observation partly explains slow transport and shorter and frequent active-runs in stressed cells. The second hypothesis, that cellular stresses induce deactivation of molecular motors, was also studied in depth. Immunofluorescence study in fixed cells showed that hyperthermia caused an immediate increase in the cytoplasmic concentration of the constitutive heat-shock-protein 70 (Hsc70), thereby detaching kinesin molecular motors from the vesicles. Furthermore, microinjection of Hsc70 in adherent cells strongly blocked active-transport in cells. This explains the predominantly diffusive transport in stressed cells. These results provide a quantitative analysis of the strong coupling between cellular stresses and intracellular transport.