Mechanisms that influence microtubule nucleation machinery localization in Drosophila melanogaster dendritic branchpoints
Open Access
- Author:
- Govindarajan, Maalavika
- Graduate Program:
- Neuroscience
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- August 08, 2023
- Committee Members:
- Melissa Rolls, Thesis Advisor/Co-Advisor
Aimin Liu, Committee Member
Sonia Cavigelli, Professor in Charge/Director of Graduate Studies
William O Hancock, Committee Member - Keywords:
- microtubule
nucleation
Drosophila melanogaster
neurons
dendritic branchpoints
cytoskeleton
cell biology
confocal microscopy - Abstract:
- Microtubules are important structural entities of the neuronal cellular milieu. While their primary functions are to maintain structural integrity and facilitate intracellular molecular transport, they also play an important role in maintaining neuronal cellular morphology, both during development and in the later stages. Microtubules (MTs) provide tracks for the molecular transport in cells, which is carried out by the motor proteins Kinesins (plus-end oriented transporters) and Dyneins (minus-end oriented transporters). MT nucleation is the process through which MTs are assembled, but it is also an important process for maintaining dendritic minus-end-out MT polarity observed in Drosophila sensory neurons. Of particular interest, is the bias of new nucleation events being directional, towards the cell body and whether it is a consequence of pre-existing MTs, which are also an important means to maintain MT polarity. Since not much is known about the processes leading up to the MT nucleation, further questions arise, like how nucleation machinery gets transported into the dendritic branchpoints, and whether the cytoplasmic Dynein heavy chain subunits dhc64c or beethoven are involved. Microtubule nucleation events are dependent on the γ-Tubulin Ring complex formation and subsequent α and β-tubulin protofilaments being attached to it. In Drosophila melanogaster, an important model for conducting genetic studies in, the branch points of the major dendritic arbor in Class 1 dendritic neurons were found to be housing alternative MT organizing centers, as opposed to the ones found usually in most cells like centrosomes (Mitchison & Kirschner, 1984), and Golgi apparatus (Miller et al., 2009; Rivero et al., 2009). Using RNAi screens in Drosophila melanogaster we studied the various molecular candidates that potentially play a role in recruiting γ-Tubulin to the site of MT nucleation in Drosophila dendrites. Our experiments led us to the conclusion that the directional bias of MT nucleation events is independent of pre-existing MT polarity, which prompts us to look into other dendritic MT effectors to understand better, in the future. We also found that more candidates and RNAi lines need to be tested before confirming the involvement of dhc64c or beethoven in γ-tubulin transport in dendritic branchpoints. Finally, we identified a candidate that has the ability to recognize membrane-curvature, Endophilin B, to also be implicated in MT nucleation, upstream to γ-tubulin, which leads us in the direction of similar molecular candidates to test, in the future. All in all, we found some preliminary information that allows us to look into MT nucleation with a clearer focus, and to eventually be able to identify all the key players in regulating these processes.