3.3.2. Temperature Block and Release of VSV-G tsO45

The envelope “G” protein of vesicular stomatitis virus (VSV-G) is a viral glycoprotein transported through the secretory pathway. VSV-G tsO45 is a thermosensitive (ts) mutant of VSV-G. VSV-G tsO45 can be expressed by transient transfection or by adenoviral transduction [38]. When synthesized at the non-permissive temperature of 40.5 °C, VSV-G tsO45 is incompletely glycosylated and misfolded, forms non-covalently associated aggregates, and is retained in the ER [39]. When cells are shifted to the permissive temperature of 32 °C, the aggregates of VSV-G tsO45 disassemble, which can then correctly fold, trimerize, exit ER, and traffic through the Golgi to the plasma membrane [38, 40]. When combined with different experimental conditions, this allows the characterization of a protein of interest in membrane trafficking. By fusing VSV-G tsO45 with fluorescent proteins, its transport from the ER to plasma membrane can be observed in real-time by live cell imaging. Given that VSV-G is N-glycosylated, a combination with endoglycosidase H (endo H) treatment can help with understanding the speed of membrane trafficking (see Subheading 3.3.4). Therefore, it has been one of the most used tools in membrane trafficking studies.

The technique of temperature block and release is frequently but not exclusively used with VSV-G O45. Temperature block and release is also used as a tool to study protein trafficking at distinct stages of the secretory pathway. By adjusting the temperature, secretory proteins can be accumulated in particular compartments in the trafficking pathway and their exit from the compartments can be synchronized. Using membrane proteins as markers, it was reported that incubation of the cells at 15 °C leads to retention of cargo proteins in the early Golgi or ER-Golgi intermediate compartment (ERGIC), while incubation at 20 °C leads to a block at the TGN [41, 42]. For example, one can first block cells at 15 °C and then shift cells to 20 °C to specifically test the speed of trafficking between the cis- and trans- Golgi under different experimental conditions [21]. The exact mechanism behind the temperature block is not fully understood, but may be related to the membrane fluidity [43] and the association of Golgi proteins such as ARF1 and ARL1 affected by the temperature change [44]. Therefore, one major drawback of this technique is that the process exposes the cells to non-physiological temperatures that affect trafficking. Moreover, a rapid and precise switch of temperature (see Note 10) is not always guaranteed and therefore variations among experiment repeats may be introduced.