Kinesin-2 and +TIP dependent directed growth maintains the uniformly polarized microtubule array in Drosophila dendrites
Open Access
- Author:
- Mattie, Floyd J
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 20, 2011
- Committee Members:
- Melissa Rolls, Dissertation Advisor/Co-Advisor
Melissa Rolls, Committee Chair/Co-Chair
Wendy Hanna Rose, Committee Member
Graham Hugh Thomas, Committee Member
Lorraine C Santy, Committee Member
Richard W Ordway, Committee Member - Keywords:
- dendrite
polarity
microtubule
Kinesin-2
+TIP
neuron
APC
adenomatous polyposis coli
EB1
noncentrosomal - Abstract:
- In many differentiated cells microtubules are organized into polarized noncentrosomal arrays, yet few mechanisms that control the formation or maintenance of these arrays have been identified. To study such arrays, I have utilized Drosophila dendritic arborization (da) neurons. These neurons have a polarized cytoskeleton that consists of two types of noncentrosomal arrays. Both the axonal and dendritic microtubule arrays consist of uniformly oriented microtubules, but the microtubules of the axon are oriented with plus-ends outward, while the microtubules of the dendrite are oriented with plus-ends inward. We have identified a novel mechanism that allows for the maintenance of uniform microtubule polarity in the branched dendrites of these highly polarized cells. All plus-ends in these dendrites grow towards the cell body. Thus, to maintain uniform microtubule polarity in dendrites, I hypothesized that the direction of microtubule growth must be controlled at branch points. Undirected growth through branch points would disrupt the uniform polarity, as growing microtubules could turn either towards or away from the cell body at each branch. To test this hypothesis, I have used whole, live Drosophila larvae for imaging growing microtubule plus-ends tagged with EB1-GFP in dendritic arborization (da) neurons. I have found that growing microtubules navigate dendrite branch points by turning the same direction 98% of the time. Observation of the plus-end behavior led to the hypothesis that growing plus-ends may be guided along pre-existing tracks of some sort. A UAS-EB1-RFP fly line was generated to test this hypothesis, and I found that the paths of growing plus-ends aligned with stable microtubule tracks. We named this phenomenon directed microtubule growth. Next, candidate proteins that may be involved in directed microtubule growth were targeted. A combination of RNAi, mutant, and dominant negative phenotypic analyses indicated that Kinesin-2 (KIF3), and the +TIPS, EB1 and APC (adenomatous polyposis coli), are required for uniform microtubule polarity specifically in dendrites. Additionally, I have investigated the extent to which dendritic geometry can compensate for disrupted directed growth. I found that in the comb dendrite of the ddaE class I da neuron uniform polarity can be more severely disrupted than in other da neurons. An analysis of the dendritic branching patterns revealed that the comb dendrite’s geometry created less of an inward bias, and thus microtubule growth was more easily randomized by disrupting the directed growth machinery. I also have shown that the Kinesin-2 subunit Kap3 can physically interact with Apc, Apc with Apc2, and Apc with EB1. Furthermore, I have found that GFP-Apc2 is highly enriched at dendritic branch points. I hypothesized that Apc would behave in the same manner, and so created UAS-GFP-Apc and UAS-RFP-Apc transgenic fly lines to test this hypothesis. Only when co-expressed with GFP-Apc2 was RFP-Apc localized to the branch points. Thus the over-expression of GFP-Apc2 can recruit RFP-Apc to branch points. We propose that Apc2 is necessary for the recruitment of Apc to dendritic branch points. At branch points, Apc is responsible for the capture of EB1 coated microtubule plus-ends, and the linkage of Kinesin-2 to the tips of growing microtubules. Kinesin-2 then steers microtubule growth along existing microtubules at branch points. This represents new functions of Kinesin-2 and +TIPs, and it is the first time the mechanism of directed microtubule growth has been observed. Currently, the Drosophila model of uniform plus-end-in microtubule polarity in dendrites conflicts with the vertebrate model of mixed (plus-end-out and plus-end-in) microtubule polarity in dendrites. It is unclear which model more accurately reflects the in vivo microtubule organization of vertebrate neurons. I have developed an experimental design that our lab will soon use to elucidate the in vivo microtubule polarity in a vertebrate. Briefly, we will use Zebrafish (Danio rerio) larvae for this investigation as they are one of the few vertebrates with neurons amenable to examination by light microscopy in whole live animals. I have described the strategy we will need to employ to successfully map the neuronal microtubule polarities of these in vivo vertebrate neurons.