Figure 3. Nmd-GFP colocalized with mitochondria at all stages of spermatogenesis. Nmd-GFP also colocalized with centrosomes early in spermatogenesis and basal bodies later in spermatogenesis. (A) In meiosis, mitochondria and Nmd-GFP are aligned with the spindle. Nmd-GFP is also at spindle poles. (B) At the onion Stage, Nmd-GFP is associated with the Nebenkern. (C) At the leaf Blade stage, Nmd-GFP is associated with unfurling mitochondria. Nmd-GFP is concentrated in the basal bodies (arrow). (D) During mitochondrial elongation, Nmd-GFP is associated with the mitochondrial derivatives and with the basal bodies (arrow).

Localization of ß-tubulin and UNC proteins during spermatogenesis in nmd deficient Drosophila melanogaster

Sarah C. Pyfrom, Samantha B. Lightcap, and Karen G. Hales Department of Biology, Davidson College, Davidson NC 28035

INTRODUCTION

Drosophila Spermatogenesis

Figure 1. Wild type spermatogenesis. Phase contrast micrographs, nuclei are phase light, mitochondria are phase dark and matching schematic diagrams. (A) Primary spermatocyte. (B) Meiosis, mitochondria aligned with spindle. (C) Mitochondria aggregate around nucleus (arrow). (D) Onion stage, mitochondria fuse to form the Nebenkern (arrow). (E) Leaf blade stage, early elongation in spermatids. (F) Late elongation, two mitochondria elongate along sperm tail sperm tail.

FUTURE WORK

ACKNOWLEDGEMENTS

Nmd-GFP colocalized with mitochondria, centrosomes, and basal bodies during spermatogenesis

During normal spermatogenesis, the mitochondria associate with the nucleus and form a tight phase-dark sphere adjacent to the nucleus (Figure 1). This sphere—formed of two interlocking mitochondrial derivatives—is called the nebenkern. The two derivatives then unfurl as the sperm assumes its mature, elongated structure.

Homozygous nmd mutant males are sterile and the nebenkern fails to form

Figure 2. Mitochondria fail to aggregate in homozygous nmdry4 males. Phase contrast micrographs of wild type (A,C), nmdry4 (B,D) and nmd2e (E-G) male spermatids. In meiosis (A,B) wild type mitochondria align with the meiotic spindle and appear as dark bars (A arrow), nmdry4 mitochondria fail to localize to the spindle and appear as dark scattered dots (B arrow). In the onion stage (C,D) wild type mitochondria fuse to form the Nebenkern (C arrow), nmdry4 mitochondria fail to align with the nucleus and remain individually dispersed (D arrow). In nmd2e males the nebenkerne frequently do not form (E), are irregularly sized/shaped (F) or contain vacuoles (G). The phenotype is rescued by a wild type transgene.

The Nmd (no mitochondrial derivative) protein, a member of the AAA ATPase superfamily, is required for mitochondrial aggregation in early post-meiotic spermatids. In nmdry4 mutant testes mitochondria fail to properly aggregate in the post-meiotic stages of spermatogenesis, resulting in male sterility. Nmd is haplosufficient and males who are heterozygous for an nmd mutation do not show the mutant phenotype.

WT

WT

Ac

B

Leaf Blade Stage

C

Elongation

D

DISCUSSION-ß-tubulin localizes distinctly between nucleus and mitochondria during onion stage in wild-type but perhaps not in mutants.-Results consistent with known localization of nmd to the centrosomes in early spermatogenesis. (Figure 3).-Mislocalization of ß-tubulin could disrupt spermatogenesis, beginning in its early stages.

-If mutant Nmd disrupts the basal body localization or formation, but does not directly affect microtubules, then it is possible that Nmd performs a different function in the basal body that is not directly related to microtubule formation or degradation.

-Perform dissections on more male nmd/6117; ß-tubulin/+ testes to better characterize ß-tubulin formation in mutants.

-Create transheterozygotes with multiple alleles of nmd to better understand the protein’s function during spermatogenesis.

-Complete crosses to create nmd/6117; UNC-GFP/UNC-GFP flies and perform dissections on testes. Visualize localization of UNC-GFP in mutants

We would like to thank Tessa Campbell, Casie Genetti, Lauren Ivey and Dr. Barbara Lom. This work was supported by Davidson College and the National Institutes of Health AREA grant

The nmd protein has recently been localized to the basal body during spermatogenesis (Figure 3). UNC is a protein that has also been localized to the centrosome and basal body. A GFP-tagged version already exists and will provide a way to visualize the position and functionality of the basal body and centrosome in nmd mutants. Similarly, ß-tubulin is a testis-specific microtubule marker for which a GFP-tagged strain of Drosophila exists.

Transgenic flies carrying a carboxyl-terminal GFP tagged version of Nmd were generated under their own endogenous promoter. Restriction sites were included on the 5’ ends of the primers for use in subsequent digestions. For minipreps, we used the appropriate Qiagen kit. Microinjections were performed by Rainbow Transgenic Flies, Inc. (Thousand Oaks, CA).

Creation of transgenes

Generation of mutant strains

The nmd hypomorphic mutant strain was originally identified in a screen described in Berg and Spradling, 1991, Genetics 127: 515-524.

Dissection and Imaging

Flies were dissected in TB1 buffer and testes/tissue were imaged using phase contrast and fluorescent confocal microscopy. Wild type flies were of the Oregon R (OR) strain.

Nmd-GFP localization Methods

BLASTing the protein sequence derived from the nmd gene sequence revealed high levels of similarity between nmd and members of the AAA+ STPase super family. Other members of this family are Spastin and Katanin. These proteins are involved in microtubule depolymerization from the minus and plus ends (respectively) of the microtubule. This gives us reason to believe that Nmd may be involved in microtubule development or degradation.

Nmd is a member of the AAA ATPase super family

Onion Stage

B

Figure5. Preliminary visualization of ß-tubulin localization during wild-type and nmd/6117 spermatogenesis. Phase contrast and fluorescence imaging of testis dissections with B-tub-GFP transgene. There are possible differences in localization between wild-type and nmd deficient localization of B-tubulin during the onion stage and elongation stage. Later stages of elongation show irregularities consistent with nmd mutation but no apparent abnormalities in B-tubulin localization or production. (A) Wild-type onion stage cells. (B) nmd/6117 onion stage cells. (C) Wild-type elongating cells. (D) nmd/6117 elongating cells.

METHODS

Fly Crosses: nmd -/- flies do not survive to adulthood, so it is necessary to make flies that are nmd/Df. This premature death is caused by other accumulated mutations associated with the nmd strain. In addition to the fly crossing scheme in Figure 4, I also created two other stocks that, when crossed, produce offspring that are nmd/Df ; UNC-GFP/TM6csb. Testis preparation: I dissected the testes from young males, then mounted them on a slide with testis buffer. I visualized the testes with a confocal microscope using phase contrast and a filter to view fluorescence in order to determine the localization of the ß-tubulin protein during all stages of spermatogenesis.

Fully Elongated Sperm

Meiosis

A

Figure 4. Fly Crossing Scheme to create nmd/6117; ß-tubulin-GFP/+ flies. The genotypes of flies used to create final stock and target genotype.

Onion Stage

Possible overrabundance and mislocalization of centrosomal ß-tubulin in nmdP{ry4} spermatids

Localization of nmd-GFP during spermatogenesis

WT

C

A

ABSTRACT The Drosophila melanogaster gene nmd codes for a protein in the AAA+ ATPase family; this family also includes known proteins spastin and katanin, which are involved in microtubule depolymerization. Visualization of GFP-tagged nmd has localized the protein to the mitochondria at all stages of spermatogenesis and with the centrosome and basal bodies during meiosis.  Males homozygous for nmd mutations are sterile or inviable in the case of complete loss-of-function of Nmd. The nmd phenotype has been characterized as a lack of mitochondrial aggregation during spermatogenesis. Rather than forming a tight and well-defined nebenkern adjacent to the nucleus, the mitochondria are scattered throughout the cytoplasm of the developing spermatocytes. The mitochondria do not elongate along the axoneme and functional, mature sperm are not present in the testis. Recent studies of the nmdP{ry4} allele show present but malformed nebenkerne during the onion stage, with frequent vacuoles or irregularities present in the mitochondrial derivative. Using fluorescence confocal microscopy, I viewed prepared mutant testes from flies with either the UNC-GFP or ß-tubulin-GFP. Preliminary visualization of GFP-tagged ß-tubulin in nmdP{ry4} homozygotes shows possible aberrancies in microtubule patterns during meiosis and early onion stages of spermatogenesis.

E F G

B

nmdP{ry4}

nmdP{ry4}

D

WT

C

ABSTRACTThe Drosophila melanogaster gene nmd codes for a protein in the AAA+ ATPase family; this family also includes known proteins spastin and katanin, which are involved in microtubule depolymerization. Visualization of GFP-tagged nmd has localized the protein to the mitochondria at all stages of spermatogenesis and with the centrosome and basal bodies during meiosis. Males homozygous for nmd mutations are sterile or inviable in the case of complete loss-of-function of Nmd. The nmd phenotype has been characterized as a lack of mitochondrial aggregation during spermatogenesis. Rather than forming a tight and well-defined nebenkern adjacent to the nucleus, the mitochondria are scattered throughout the cytoplasm of the developing spermatocytes. The mitochondria do not elongate along the axoneme and functional, mature sperm are not present in the testis. Recent studies of the nmd2e allele show present but malformed nebenkerne during the onion stage, with frequent vacuoles or irregularities present in the mitochondrial derivative. Preliminary visualization of GFP-tagged ß-tubulin in nmd2e homozygotes shows possible aberrancies in microtubule patterns during meiosis and early onion stages of spermatogenesis.

Localization of ß-tubulin and UNC proteins during spermatogenesis in Nmd deficient Drosophila melanogaster