Investigating the role of BPAG1 in axonal transport
- Mridu Kapur.
- Feb. 2013.
- Physical description
- online resource (xi, 135 pages) : illustrations (some color)
- Includes bibliographical references (p. 116-135).
- The intracellular trafficking of vesicles and organelles is essential for all cells. Neurons are especially reliant on cargo transport as a consequence of their extremely polarized morphology. Highlighting this point, disruption of axonal transport has been attributed to many neurodegenerative diseases. In recent years, tremendous progress has been made towards a better understanding of the fundamental elements of axonal transport. However, the regulatory mechanisms that control its dynamics remain largely unknown. One striking feature of the axonal transport system is that the retrograde transport of endosomes within axons proceeds with remarkable speed and uniformity, despite having to navigate a complex and crowded environment. In this dissertation, I propose a role for BPAG1n4, a massive 600kDa protein, in the regulation of the processivity of axonal transport. BPAG1n4 has previously been shown to play an essential role in retrograde axonal transport by virtue of its interactions with the dynactin subunit p150Glued, and with endosomes via retrolinkin. Its ability to interact with the main components of the transport system make it ideally placed to coordinate retrograde transport. Here, I show that BPAG1n4 interacts with EB1 family proteins at the microtubule plus end. Using single molecule labeling coupled with live imaging, I found that expression of the isolated plus end binding domain results in a substantial impairment in the processivity of retrograde axonal transport in dorsal root ganglion (DRG) neurons. This suggests that the accessibility of the microtubule plus end to proteins such as BPAG1n4 is essential for maintaining the processive motility of endosomes along the axon. Interestingly, I have discovered an extremely rapid, calcium regulated switch between microtubule plus end interaction and lattice binding in the carboxyl terminus of BPAG1n4. This novel, calcium dependent regulatory mechanism is EF-hand dependent, and mutations of the EF-hands abolish this dynamic behavior. This study represents the first evidence of calcium signaling directly modulating the dynamic interaction of a protein with microtubules. Strikingly, I observed that disruption of the calcium responsiveness of BPAG1n4's association with the plus end severely impaired processive endosomal transport, suggesting the exciting possibility that calcium may play a role in regulating the traffic of endosomes along axons. This study thus provides exciting new insight into the molecular mechanisms that regulate the complex system of axonal transport. This domain of BPAG1n4 is conserved in other members of the spectraplakin family, suggesting that calcium regulation of microtubule plus end interaction may play a vital role in their functional activities as well. Addressing the potential role of this calcium switch may be crucial to fully understanding the diverse functional activities of spectraplakins in microtubule based cellular processes.
- Publication date
- Submitted to the Program in Neurosciences and the Committee on Graduate Studies of Stanford University.
- Thesis (Ph.D.)--Stanford University, 2013.