Date of Award
Doctor of Philosophy (PhD)
Adam D. Linstedt
The mammalian Golgi ribbon is a highly dynamic organelle, notoriously difficult to study and interpret. It is a nexus of the endomembrane system, with entry and exit at both ends of the stack. Cargo typically transports through the Golgi on the order of 20 minutes. However, traditional methods of cell biology used to determine the function of a protein through knockouts and knockdowns, result in aberrant end states that may be far removed from the initial functions. Further complicating efforts is that the Golgi undergoes complex rearrangements in response to the cell cycle, stress, development, and migration. Defects in a complex, interconnected membrane system at one level of the pathway can quickly cascade and compound into other secondary effects. Proper study requires analysis of the period immediately following loss of the protein of interest.One approach that allows for this is acute inactivation. Unfortunately, this tool has been mostly of limited use in mammalian cells as selectively targeted drugs and temperature sensitive mutants are difficult to come by. Instead, we have begun using a technique called Chromophore Assisted Light Inactivation (CALI) to acutely inactivate proteins. Though the practice and concepts of CALI have been around for the past 25 years, it has remained underused due to technical limitations and hazardous materials. Recently with the advent of KillerRed, a genetically encoded photosensitizer, we have been able to temporally control inactivation of specific protein complexes and observe the cell's response within the initial minutes post inactivation. Here we use KillerRed to address questions surrounding two different steps in Golgi biogenesis, ribbon formation and endoplasmic reticulum export.
Initially, we described the methods and effectiveness of using KillerRed tagged proteins to inactivate complexes of endogenous proteins in trans by expressing KillerRed tagged sec13, a component of the COPII coatomer which is required for vesicular trans- port from the endoplasmic reticulum. Irradiation of sec13-KillerRed expressing cells blocked export of cargo and Golgi proteins from the endoplasmic reticulum, consistent with long term inhibition experiments. Remarkably, the acute block of endoplasmic reticulum exit resulted in the distribution of early-but not late-Golgi residents to peripheral punctae that correspond to the endoplasmic reticulum-Golgi intermedi ate compartment. These results identify a omnipresent recycling pathway required for maintenance of the Golgi ribbon. Further, the return to the Golgi requires input from the endoplasmic reticulum.
Subsequently, we dissected the different roles that two mammalian Golgi tethers, GRASP65 and GRASP55, play in maintenance of the Golgi ribbon using a combination of KillerRed inactivation with fluorescence recovery after photobleaching to measure Golgi ribbon integrity at discrete levels of the Golgi stack. Inhibition of GRASP65 which resides on the cis-Golgi, results rapid loss of fluorescence recovery after pho- tobleaching of cis- but not trans-Golgi residents. GRASP55 inhibition resulted in a rapid loss of trans- but not cis-Golgi resident recovery. The distinction of duties was functionally significant, as rescuing the loss of GRASP65 from the cis-Golgi with redistributed GRASP55 restored integrity of the ribbon but sacrificed compartmentalization and proper glycosylation of exported cargo. These data identify GRASPs as novel regulators of Golgi subcompartmentalization identity. Thus the division of duties among two GRASPs in Golgi ribbon forming cells is important for proper processing of cargo.
Collectively, the use of KillerRed has given evidence to old predictions as well been useful in framing more traditional molecular biology experiments.
Jarvela, Timothy S., "Acutely controlled inactivation reveals requirements for maintenance and subcompartmentalization of the mammalian Golgi ribbon" (2013). Dissertations. 281.