Dr. Davie Van Vactor

Axon Guidance in Drosophila

Lowery LA, Lee H, Lu C, Murphy R, Obar RA, Zhai B, Schedl M, Van Vactor D, Zhan Y. 2010. Parallel genetic and proteomic screens identify Msps as a CLASP-Abl pathway interactor in Drosophila. Genetics. 185(4): 1311-25.

Lowery LA and Van Vactor D. The trip of the tip: understanding the growth cone machinery.
Nat Rev Mol Cell Biol. May 2009. 10(5): 332-43.

Question 1

Can each of you briefly tell us a bit about yourself, your career path over the years, and specifically what brought you to begin working on axon guidance in Drosophila?

Michael J. F. Barresi

Question 2

Part 1. I feel that in a lot of the research, you need to fight for every little piece of data. However, in your approach generated such a large amount of data that it seems overwhelming to me. I am wondering how you approached this data to organize and filter through it to pick the ones of your interest?

Part 2. Can you specify the importance of using coupled genetic and proteomic screening approach in this research? It seems that it does not always give us identical results. You mentioned in the paper that the presence of Zipper was prevalent in proteomic screen, but only showed mild interaction with CLASP in genetic screen. Are there other examples like this? What would be the possible explanation?

Yisi Lu

Question 3

In the paper, “Parallel Genetic and Proteomic Screens Identify Maps as a CLASP-Abl Pathway Interactor in Drosophila,” it states that “While some of the genetic loci showed the same type of interaction (suppression or enhancement [of CLASP]) with both the Df and transposon alleles (in 17 cases), 9 showed opposite effects” (1315).

I understand this result could be explained by the theory that the transposon insertions are in or near the 5’-UTR of the loci, which contain a UAS element with the correct sequence to initiate GAL4-UAS-dependent GOF; however, if loci are acting as both enhancers and repressors, how can we definitively relate them to specifically to CLASP expression? Also, what did the phenotypes of these nine cases reveal? Were they similar to the wild type?

Samantha Torquato

Question 4

I found a paper published in 2003 [Kramer and Staveley, 03. Gal4 causes developmental defects and apoptosis when expressed in the developing eye of Drosophila melanogaster. Genet Mol Res. 2(1)43-7.] that showed that the GMR-GAL4 expression system has an effect on drosophila eye development:
GMR-GAL4 homozygotes have a highly disorganized ommatidial array and levels of apoptosis in the third star larval eye imaginal disc are higher compared to wt flies. Also, these morphological defects are significantly enhanced when flies are raised at 29C, which incidentally corresponds very close to the temperature at which abl GOF and msps GOF disrupt retinal development (29.5C). Is it possible that the GMR-GAL4 expression system itself could be causing some of the eye defects observed thereby distorting the results of the screen and the abl/msps GOF phenotype? How would you alter/test the expression system to accommodate these findings and ensure the results are completely controlled?

Yvanka De Soysa

Question 5

CLASP and Msps antagonized each other because the defect from msps LOF was exacerbated by CLASP GOF and vice versa. Then is there any test done for LOFs for both genes or GOFs? How do you predict the result?

Wenxin Cai

Question 6

In the article, “Parallel Genetic and Proteomic Screens Identify Msps as a CLASP-Abl Pathway Interactor in Drosophila” it states that CLASP binds to IGGAP1 which is an actin binding protein, but in the drosophila this protein is not present. You suggested that in place of this there might be other interactions involved in how CLASP links MTs and actin. What are the other ways this may occur?

Charmaine Rivera

Question 7

You stated that Cam and Abl work synergistically in the Drosophila embryonic CNS to control midline axon guidance, and that msps mutants show axon guidance defects, did you test axon guidance in a double a mutant for Cam and msps LOF?

Zarnab Iftikhar

Question 8

In figure 5E, you show that some msps GOF mutant embryos had defects in fascicle morphology and/or ectopic fascicle crossing. What might explain why (1) not all mutants showed defects and (2) why not all GOF mutants with defects showed the same kinds of defects?

Danielle Vazquez

Question 9

Why would the repellent effects of Slit on cone growth orientation be mitigated when CLASP and Abl are mutated? And how do you propose that Msps modulates slit repulsion?

Rachael Stein

Question 10

The paper mentions the necessity for a balance of turning responses to attractive Netrin and repellent Slit and this balance is possible because of the molecular mechanisms of CLASP and Msps. Can you elaborate on the balance of response to both Netrin and Slit and how it influences axon guidance? And specifically, how has Abl been shown to mediate both Netrin and Slit?

Mary Roh

Question 11

The paper stated that, “CLASP has not previously been linked with translation/RNA regulation, but the identification of these two genes points to an intriguing possibility of crosswalk between cytoskeletal regulations and translations,” what further study would you do to the CLASP to show clearly that the two genes can possibly crosswalk? What is your possible hypothesis?

Hyunkyung Kang

Question 12

The introduction of localized adhesive cues leads to an increase in the number of exploratory MTs that interact with the adhesion site in the P domain. Does a relationship exist between the strength of cues and the number of additional exploratory MTs recruited?

Julie Wang

Question 13

I find it curious that so many developmental milestones seem similar in their processes. Do you think there are any similarity in the cytoskeletal processes involved in Primordial Germ Cell guidance or any other migrating cell for that matter with the processes involved in axon guidance?

Carla Velez

Question 14

Currently in your opinion what are the most pressing questions in the study of Axon guidance, and what steps is your lab taking to address these questions?

Michael J.F. Barresi