Download 00:40, 26 August 2010

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
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.
INTRODUCTION
Drosophila 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.
Localization of nmd-GFP during
spermatogenesis
Meiosis
Possible overrabundance and mislocalization of
centrosomal ß-tubulin in nmdP{ry4} spermatids
Onion Stage
WT
A
A
Onion Stage
nmdP{ry4}
B
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.
Homozygous nmd mutant males are sterile and the
nebenkern fails to form
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.
Leaf Blade Stage
F
WT
C
WT
Elongation
nmdP{ry4}
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).
METHODS
Nmd is a member of the AAA ATPase super family
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.
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.
Nmd-GFP localization Methods
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.
Creation of transgenes
Transgenic
flies carrying a carboxyl-terminal GFP tagged version of Nmd
B
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).
D
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 Btubulin 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.
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.
Nmd-GFP colocalized with mitochondria, centrosomes,
and basal bodies during spermatogenesis
Ac
C
C
G
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.
Fully Elongated Sperm
WT
D
E
B
FUTURE WORK
-Perform dissections on more male nmd/6117; ß-tubulin/+
testes to better characterize ß-tubulin formation in
mutants.
Figure 4. Fly Crossing Scheme to create nmd/6117; ß-tubulinGFP/+ flies. The genotypes of flies used to create final stock and
target genotype.
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.
-Create transheterozygotes with multiple alleles of nmd to
better understand the protein’s function during
spermatogenesis.
-Complete crosses to create nmd/6117; UNC-GFP/UNCGFP flies and perform dissections on testes. Visualize
localization of UNC-GFP in mutants
ACKNOWLEDGEMENTS
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
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 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