UNC-33 and RPM-1 Function in Parallel to Regulate Axon Termination

Rayna Birnbaum

A Thesis Submitted to the Faculty of

The Wilkes Honors College

in Partial Fulfillment of the Requirements for the Degree of

Bachelor of Arts in Liberal Arts and Sciences

with a Concentration in Neuroscience

UNC-33 AND RPM-1 FUNCTION IN PARALLEL TO REGULATE AXON TERMINATION

by Rayna Birnbaum

This thesis was prepared under the direction of the candidate’s thesis advisors, Dr. Paul Kirchman and Dr. Brock Grill, and has been approved by the members of her supervisory committee. It was submitted to the faculty of The Honors College and was accepted in partial fulfillment of the requirements for the degree of Bachelor of Arts in Liberal Arts and Sciences.

SUPERVISORY COMMITTEE:

______Dr. Paul Kirchman

______Dr. Brock Grill

______Dean Jeffrey Buller, Wilkes Honors College

______Date

ii ABSTRACT

Author: Rayna Birnbaum

Title: UNC-33 and RPM-1 Function in Parallel to Regulate Axon

Termination

Institution: Harriet L. Wilkes Honors College of Florida Atlantic University

Thesis Advisor: Dr. Paul Kirchman

Degree: Bachelor of Liberal Arts and Sciences

Concentration: Neuroscience

Year: 2016

Precise axon termination is necessary for the development of a functioning neuronal network within the nervous system. However, little is known about the mechanisms that regulate axon termination. C. elegans RPM-1, a conserved member of the PHR proteins, has been previously shown to regulate axon termination and synapse formation. Recently, it was shown that, when phosphorylated by Cdk5, CRMP-2 (the mammalian homolog of

C. elegans UNC-33) acts as a microtubule destabilizer during axon outgrowth. We investigated the relationship between the RPM-1 and UNC-33 pathways in axon termination. Our data has lead to the conclusion that the CDK-5, UNC-33 pathway works in parallel with the RPM-1 pathway to regulate axon termination.

iii TABEL OF CONTENTS

INTRODUCTION ………………………………………………………………… Page 1

MATERIALS AND METHODS ………………………………………………….. Page 2

RESULTS …………………………………………………………………………. Page 3

DISCUSSION ……………………………………………………………………... Page 8

LIST OF FIGURES

Figure 1: RPM-1 candidate screen ………………………………………………… Page 5

Figure 2: Analysis of genetic relationships between cdk-5, unc-33 and rpm-1……. Page 7

Figure 3: RPM-1 and UNC-33 function in parallel to regulate axon termination .. Page 10

iv INTRODUCTION

The development of a functional neuronal network relies upon each individual neuron reaching its appropriate target before stopping growth and forming a synapse. For this very specific action to be achieved accurately, the axon must extend through the environment and terminate in its correct anatomical position. Once the axon has reached its appropriate anatomical position, the growth of the axon stops, a process referred to as axon termination. Currently, there is relatively little known about the mechanisms that regulate the process of axon termination.

Caenorhabditis elegans Regulator of Presynaptic Morphology 1 (RPM-1) is a conserved member of the PHR proteins, including Drosophila Highwire and human PAM

(MCYBP2). The PHR proteins regulate neuronal development through E3 ubiquitin ligase activity, which is conserved across species (Po et al. 2010). The RPM-1 ubiquitin ligase activity is functional when RPM-1 forms a complex with the F-box protein, FSN-1

(Liao et al. 2004). Loss-of-function mutations in rpm-1 have been shown to cause defects in axon termination and synapse formation in the mechanosensory and motor neurons

(Grill et al. 2016).

Axon outgrowth is dependent upon the ability of an axon to precisely navigate through its environment. Axon navigation occurs via dynamic changes in the cytoskeleton in response to repulsive or attractive extracellular guidance cues (Tassier-Lavigne &

Goodman 1996). The cytoskeletal reorganization induced by microtubule-associated proteins (MAPs) can cause stabilization or destabilization of microtubule dynamics (Dent

1 & Gertler 2003). Microtubule dynamics are regulated by attractive and repulsive guidance cues, which cause stabilization and outgrowth, or destabilization and reorientation, respectively (Buck & Zheng 2002).

Mammalian Collapsin Response Mediator Protein (CRMP-2), the homolog of C. elegans

UNC-33, has been shown to promote microtubule assembly during axon outgrowth by assisting in tubulin binding (Fukata et al. 2002). Recent data has shown that the repulsive guidance cue Semaphorin3A functions through the intracellular protein CRMP-2

(Goshima et al. 2000; Brown et al. 2004). Within the Sema3A signaling cascade, when phosphorylated by Cyclin-Dependent Kinase 5 (Cdk-5) and glycogen synthase kinase 3β

(GSK-3β) CRMP-2 has reduced tubulin affinity inducing microtubule destabilization

(Uchida et al. 2005; Yamashita et al. 2007; Yoshimura et al. 2005)

Here, we wanted to investigate the relationship between RPM-1 and the UNC-33 pathway in axon termination. Using genetic manipulation, we show that CDK-5 and

UNC-33 function within the same pathway and in parallel to RPM-1 to regulate of axon termination.

MATERIALS AND METHODS

Alleles used in this study include the following: fsn-1(gk429), cdk-5(ok626), rpm-1(ju44), and unc-33(e204). All alleles cause a loss of function. Double and triple mutants were constructed following standard C. elegans mating procedures and kept at a temperature of

23° C. PCR (Polymerase Chain Reaction) genotyping was used to confirm all mutant

2 strains generated. Analysis of axon termination was done using the following fluorescent transgenes: muIs32[Pmec-7GFP] and zdIs5[Pmec-4GFP]. The muIs32 transgene was used for the initial screen with fsn-1, and zdIs5 was used to analyze relationships with rpm-1. The mutant hooking phenotype in the rpm-1; muIs32 mutants are approximately 90% penetrance, making enhancer effects difficult to detect. Therefore, we used the zdIs5 transgene because the rpm-1; zdIs5 mutants have approximately 20-40% penetrance of hooking phenotype, allowing for identification of enhancer effects.

The mechanosensory neurons are used to study axon termination because of their specific anatomical termination under wild-type conditions (Schaefer et al. 2000). For the analysis of axon termination defects in different mutants, live imaging was conducted using adult animals. Live animals were anesthetized using Levamisole (5nM) in M9. The imaging was conducted using an epiflourescent microscope (Leica CFR5000) with an oil- immersion lens at 40X magnification and a CCD camera (Leica DFC345 FX).

RESULTS

To assess the function of a given gene within the C. elegans model system, axon termination defects are categorized and quantified. A promoter specific to the mechanosensory neurons was used to transgenically express the fluorescent protein GFP.

We chose to examine axon termination defects using the mechanosensory neurons because of their specific axon termination sites in the wild-type animal. The PLM axon runs from the cell body in the posterior end of the animal and extends out towards the anterior, terminating prior to the ALM cell body in the middle of the animal (Fig. 1A). In

3 rpm-1 and fsn-1 mutants, axon termination defects of the PLM neurons manifest in an overextension phenotype and a hooking phenotype (Fig. 1A; Grill et al., 2007). The overextension phenotype occurs when the axon extends past the ALM cell body before terminating, and the hooking phenotype occurs when the axon extends past the ALM cell body and hooks ventrally before terminating.

During a candidate screen for proteins working within the RPM-1 pathway to regulate axon development, a cdk-5; fsn-1 double mutant was created. fsn-1 encodes for the F-box protein that functions in a ubiquitin ligase complex with RPM-1 (Liao et al. 2004). The frequency of axon termination defects in cdk-5; fsn-1 double mutants significantly enhanced with 37 ± 3.6% hooking defects (Fig. 1B). This indicated that CDK-5 was working in a parallel pathway to FSN-1 to regulate axon termination. Because previous studies have shown CDK-5 functions within the UNC-33 pathway, we wanted to investigate the relationship between RPM-1, CDK-5, and UNC-33 in axon termination.

4 A

wt fsn-1 fsn-1

ALM PLM

Overextension Hooking

B % PLM Termination Defect 100

80 *** 60 *** Overextension 40 Hook

0 wtWT fsnfsn-1 -1 cdkcdk-5 -5 cdk-5; fsn-1 cdk-5; fsn-1

Figure 1: RPM-1 candidate screen. A. Anatomical Caenorhabditis elegans representation adapted from Grill et al., 2007. The blue box indicated the anatomical region of the animal presented in the following images. The transgene muIs32[Pmec-7GFP] was used to visualize the PLM axon. In wild-type animals, the PLM axon terminates prior to the ALM cell body. Overextension phenotype in an fsn-1 mutant, in which the PLM axon fails to terminate and overextends past the ALM cell body. Hooking phenotype in an fsn-1 mutant in which the PLM axon hooks prior to termination. B. Quantification of the PLM axon termination defects, showing the averages of 5-6 counts for each genotype (30-40 neurons/count). Significance was determined using a Student’s t test. ***, p < 0.001.

To begin, we crossed the cdk-5 mutant with an rpm-1 mutant to determine if an enhancer

effect would occur in this double mutant as well. Similar to the cdk-5; fsn-1 double

mutant, the cdk-5; rpm-1 double mutant had a 36 ± 4.5% PLM hooking phenotype,

5 indicating a mild enhancer effect (Fig. 2B). Because previous studies show CDK-5 is an upstream regulator of UNC-33 in cultured cells, the cdk-5; unc-33 double mutant was created. The double mutant resulted in a 9 ± 1.6% hooking phenotype, which was not significantly different compared to the 4-6% hooking phenotype of single mutants, indicating a lack of enhancer effect (Fig. 2B). Next, the triple mutant of all three genes, cdk-5, unc-33, and rpm-1, was generated. Off this triple mutant cross, all double mutants and single mutants were also isolated. Data for double and single mutants consistent with our previous data collected, indicating a lack of background mutations that could have caused misleading results. The unc-33; rpm-1 mutant had a strong enhancer effect with

75 ± 4.8% hooking phenotype (Fig. 2B). The cdk-5; unc-33; rpm-1 mutant showed the same degree of enhancement in the axon termination defects as the unc-33; rpm-1 mutant, a 74 ± 4.8% mutant hooking phenotype in the PLM neurons (Fig. 2B).

wt rpm-1 rpm-1 ALM

PLM Overextension Hooking

Overextension B % PLM Termination Defect Hook

100 ns *** 80 ns ns 60 * 40

0 WT wt cdkcdk-5 -5 unc-33 unc-33 rpmrpm-1 -1 cdk-5; rpm-1 cdk-5; cdk-5; cdk-5; unc-33; unc-33; cdkcdk-5; -5; rpm-1 unc-33 rpm-1 unc-33 rpm-1 uncunc-33; -33; rpmrpm-1 -1

Figure 2: Analysis of genetic relationships between cdk-5, unc-33 and rpm-1. A. Anatomical Caenorhabditis elegans representation adapted from Grill et al., 2007. The blue box indicates the anatomical region of the animal presented in the following images. The transgene zdIs5[Pmec-4GFP] was used to visualize the PLM axon. In wild-type animals, the PLM axon terminates prior to the ALM cell body. Overextension phenotype in an rpm-1mutant, in which the PLM axon fails to terminate and overextends past the ALM cell body. Hooking phenotype in an rpm-1 mutant in which the PLM axon hooks prior to termination. B. Quantification of the PLM axon termination defects for the genetic crosses to determine the relationships between cdk-5, unc-33 and rpm-1. Showing the averages of 5-6 counts for each genotype (30-40 neurons/count). Significance was determined using a Student’s t test. ***, p < 0.001; *, p < 0.05; ns, not significant.

7 DISCUSSION

Axon termination needs to be accurate for the formation of a functioning neuronal network. Previously, certain genetic pathways have been implicated in the execution of axon termination, including the RPM-1 pathway within the C. elegans model. The UNC-

33 pathway, functioning through phosphorylation of UNC-33 via CDK-5, plays a role in microtubule destabilization (Uchida et al. 2005). Our results indicate that the CDK-

5/UNC-33 pathway is also needed for successful axon termination, possibly because of impacts on growth cone collapse (Uchida et al. 2005; Yamashita et al. 2007; Yoshimura et al. 2005).

Because of the mild enhancement in fsn-1; cdk-5 mutants, I concluded that CDK-5 works in a parallel pathway to FSN-1. This could occur because CDK-5 is parallel to the RPM-1 pathway as a whole, or parallel to FSN-1 within the RPM-1 pathway.

Further genetic comparison showed that the cdk-5; rpm-1 double mutant is enhanced.

This indicates that CDK-5 is working in parallel to the RPM-1 pathway, and not within the RPM-1 pathway. A lack of enhancer effects in cdk-5; unc-33 double mutants leads to the conclusion that CDK-5 and UNC-33 are working within the same pathway, consistent with prior results in cultured cells. The unc-33; rpm-1 mutant has a very strong enhancer effect, which highlights the parallel relationship between RPM-1 and UNC-33 in axon termination. The almost identical strong enhancer effects seen in the unc-33; rpm-1 mutant and the cdk-5; unc-33; rpm-1 triple mutant supports my overall hypothesis that

8 the CDK-5/UNC-33 pathway works in parallel to the RPM-1/FSN-1 pathway to regulate axon termination (Fig. 3).

CDK-5 has been shown to phosphorylate UNC-33 and induce microtubule destabilization through the Semaphorin3A signaling cascade, which is thought to cause growth cone collapse (Schmidt & Strittmatter 2007). My results are consistent with the UNC-33 pathway regulating axon termination by affecting growth cone collapse. The regulation of axon termination through the CDK-5/UNC-33 pathway is a novel finding that could lead to greater understanding of the axon termination process.

Because the enhancer effect seen in the cdk-5; rpm-1 mutant is so mild compared to enhancer effect of the unc-33; rpm-1 mutant, it is suggestive of possible other players within the UNC-33 cascade. Because GSK-3β has been implicated as another kinase that phosphorylates CRMP-2 (Uchida et al. 2005; Yamashita et al. 2007; Yoshimura et al.

2005), the relationships between CDK-5, UNC-33 and the C. elegans homolog to GSK-

3β, GSK-3, should be investigated. If the previous findings in cultured cells are supported in the in vivo C. elegans model system, the triple mutant between cdk-5, rpm-1 and the C. elegans homolog to GSK-3β should result in a defect phenotype similar to that of the unc-33; rpm-1 mutant and the cdk-5; unc-33; rpm-1 mutant. As well, the extracellular regulators of RPM-1 have yet to be identified; hence, an investigation into the

Semaphorin3A pathway as a potential regulator of RPM-1 may be beneficial. While my experiments did not identify any molecules that function within the RPM-1 pathway, my work did assist in elucidating the signaling networks that regulate axon termination.

9 CDK-5

RPM-1 UNC-33

Axon Termination

Figure 3: RPM-1 and UNC-33 function in parallel to regulate axon termination. The lack of an enhancer effect in cdk-5; unc-33 mutants indicates that UNC-33 and CDK-5 work within the same pathway. Previous data suggests that CDK-5 works upstream of UNC-33 (Uchida et al. 2005). The similar strong enhancer effects seen in unc-33; rpm-1 mutants and cdk-5; unc-33; rpm-1 mutants suggests that the UNC-33 pathway works in parallel to the RPM-1 pathway to regulate axon termination.

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