A Prolactin-Inducible T Cell Gene Product Is Structurally Similar to the Aspergillus nidulans Nuclear Movement Protein NUDC
Shelli M. Morris,
Paul Anaya,
Xin Xiang,
N. Ronald Morris,
Gregory S. May and
Li-yuan Yu-Lee
Department of Cell Biology (S.M.M., P.A., G.S.M.,
L-y.Y-L.) Department of Microbiology and Immunology (L-y.Y-L.)
Department of Medicine (L-y.Y-L.) Baylor College of Medicine
Houston, Texas 77030
Department of Pharmacology (X.X.,
N.R.M.) University of Medicine and Dentistry of New Jersey
Robert Wood Johnson Medical School Piscataway, New Jersey 08854
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ABSTRACT
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Clone 15 (c15) was
originally identified as a PRL-inducible gene in activated T cells.
Sequence analysis of c15 revealed that the last 94 amino
acids of c15 are 68% identical and 78% similar to the filamentous
fungus Aspergillus nidulans nuclear movement protein NUDC.
The identification of the mammalian (rat) c15 protein suggests that the
carboxy-terminal NUDC-like region has been conserved over evolution for
an important structure and/or function. To determine whether c15 is
functionally analogous to NUDC, complementation studies were performed
using the inducible/repressible pAL5 vector system. The results of the
complementation experiments show that the full-length mammalian c15
protein is not only capable of rescuing the nuclear movement defect of
the nudC3 mutants, but is also able to restore the
expression of the downstream endogenous NUDF protein to near wild type
levels. These results indicate that rat c15 and fungal NUDC not only
share similar structures, but also serve similar functions. Taken
together, the structural and functional conservation between c15 and
NUDC is consistent with the notion that c15 is the rat
homolog of nudC and has therefore been given the name
RnudC.
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INTRODUCTION
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When T cells come in contact with antigen-presenting cells or
antigen-bearing target cells, engagement of cell surface receptors
results in the expression of a number of activation genes that together
set the genetic program leading to T cell proliferation,
differentiation, or apoptosis (1, 2). In addition to the induction of
gene expression in activated T cells, another early activation event
involves a massive reorganization and polarization of the cytoskeleton
that reorients the centrosome microtubule-organizing center (MTOC), its
associated microtubules (MTs), and the Golgi apparatus toward the site
of cell contact (3). This reorganization optimally positions the
cells MT-dependent vesicle transport system in alignment with the
target cell to serve directed protein transport between cells, an
important feature of T cell function. Understanding how T cells
correctly position their nuclei and associated organelles during
activation and proliferation is a critical step to a better
understanding of the overall life cycle and effector functions of T
cells.
Reorientation and movement of intracellular organelles, in particular,
the centrosome MTOC, the nucleus, and the Golgi, depends on MT
interactions with the cell cortex and is coordinated by forces acting
on the centrosomes themselves (4, 5, 6). Perhaps the best evidence that
there are forces acting directly on the centrosomes comes from studies
of embryogenesis in Drosophila melanogaster. In the
Drosophila embryo, nuclei migrate to the cortex before
cellularization (7). If nuclear division is aborted, either by drug
action or mutation, the centrosomes nevertheless continue to multiply
and migrate to the cortex independently of the nuclei (8, 9). This
provides strong evidence that nuclear migration is mediated through a
force acting directly on the centrosome itself, which in turn serves to
move the nucleus.
In simple eukaryotes such as fungi and yeast, nuclear movement is a
MT-dependent process (10, 11). The main MT-dependent "motor"
involved in centrosomal and nuclear positioning has been identified as
cytoplasmic dynein. The evidence that cytoplasmic dynein is the main
motor protein for nuclear positioning comes from the studies of the
nudA, ro-1, and DYN1 genes, which encode the cytoplasmic
dynein heavy chains of Aspergillus nidulans, Neurospora
crassa, and Saccharomyces cerevisiae, respectively.
Mutations in the nudA gene and the ro-1 gene
cause a failure of nuclear movement through the germ tubes (12, 13). In
A. nidulans, the dynein heavy chain has been localized to
the tip of the growing germ tube (14). A mutation in the 8-kDa A.
nidulans dynein light chain, encoded by the nudG gene,
causes the heavy chain to be lost from the tip and also affects nuclear
movement (15). Similarly, disruption of DYN1 causes a partial failure
of nuclear segregation between mother and daughter cells in yeast. This
failure to segregate is caused by a defect in the ability of mitotic
nuclei to migrate to the correct orientation before anaphase (16, 17).
Additionally, in yeast, MTs attached to the spindle pole body (SPB),
the centrosome equivalent in lower eukaryotes, have been shown to be
essential for the nuclear orientation process (18). Furthermore,
several of the N. crassa ropy mutations have been shown to
affect dynactin, a complex that is required for the coupling of dynein
to its cargo, and result in a nuclear movement defect (13, 19, 20). In
yeast, mutations have also been identified that affect nuclear
positioning (21, 22, 23). Together these data support a model in which SPB
orientation and nuclear movement are mediated via an interaction
between SPB MTs and a dynactin-associated, minus end-directed dynein
motor located at the cell cortex.
In filamentous fungi, e.g. A. nidulans and
N. crassa, nuclear movement is required for normal
colony formation (13, 24). During the germination of conidia (asexual
spores), the parental nucleus divides and the daughter nuclei move out
in the germ tube of the developing germling. Temperature-sensitive
mutations have been identified in A. nidulans that affect
this initial stage of nuclear movement as well as A.
nidulans ability to maintain nuclei of additional nuclear
divisions at a uniform distance from each other. Because of the
observable defect in nuclear distribution,
this class of mutants was call nuds (24). In addition to
nudA and nudG, two other genes have been
identified, nudC and nudF (12, 24, 25, 26). The
nudC gene encodes a 22-kDa protein of unknown function (25).
The nudF gene encodes a 49-kDa protein that is similar to
the human lissencephaly (LIS-1) protein (26). LIS-1 is involved in
controlling neuronal migration in the cerebral cortex (27). Through
A. nidulans studies, NUDC has been shown to
posttranscriptionally regulate NUDF (26). Additionally, NUDF appears to
be an upstream regulator of cytoplasmic dynein/dynactin function, as a
newly discovered mutation in the cytoplasmic dynein heavy chain acts as
a bypass suppressor of the nudF deletion (D. A. Willins and
N. R. Morris, unpublished data). Therefore, through genetic studies,
NUDC can be placed upstream of NUDF, and in turn both NUDC and NUDF can
be placed upstream of dynein and dynactin.
The rat Nb2 T cell line can be stimulated to proliferate by the
addition of 110 ng/ml PRL to the cell culture medium (28). One of the
PRL-inducible genes cloned from Nb2 T cells is clone 15
(c15) (29). The c15 gene encodes a 332-amino acid
(aa) protein (45-kDa) in which the carboxy-terminal 94 aa (Gly 239 to
Asn 332) are 68% identical to the carboxy-terminal portion of the
A. nidulans nuclear movement protein NUDC (Gly 105 to Gly
198). This striking similarity suggests that the dynein/dynactin
pathway that mediates nuclear movement in fungi may also be involved in
reorientation of the centrosomal MTs and Golgi in T cells, and that the
c15 and nudC gene products may share similar
functions. The present work was designed to test the hypothesis that
c15 and NUDC are functionally related by determining whether the rat T
cell c15 protein could complement the temperature sensitivity of the
A. nidulans nudC3 mutation.
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RESULTS
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c15 Complementation Strategy and Constructs
The temperature-sensitive A. nidulans used in this
study contain the pyrG89 mutation. These A.
nidulans lack orotidine 5'-P decarboxylase, an enzyme required for
the uracil and uridine synthetic pathway (30). Therefore, to grow, the
pyrG89 A. nidulans require exogenous uracil and uridine in
their growth media. The pAL5 vector was generated by cloning the
A. nidulans histone H2A 3'-fragment polyadenylation site
downstream of the pAL3 multicloning site (Fig. 1
) (31, 32). The H2A
3'-fragment allows for more efficient processing and export of the
mature mRNA from the nucleus and greater stability of the mRNA
transcript. The pAL5 vector contains, as does the pAL3 parental vector,
the alcA alcohol dehydrogenase I gene promoter, which is
inducible or repressible based on the growth media and carbon source
present. Transcription of the gene of interest is repressed when
A. nidulans containing this construct are grown on rich
glucose media (MAG or YAG), and expression is allowed when A.
nidulans are grown on glycerol-containing media (32, 33). In
addition, both pAL5 and pAL3 vectors contain the ampicillin resistance
gene, to allow for selection in E. coli, and the N.
crassa pyr4 gene. The pyr4 gene encodes
for orotidine 5'-P decarboxylase and is capable of complementing the
pyrG89 mutation in A. nidulans, thereby allowing
A. nidulans to grow in the absence of exogenous uracil and
uridine (30).

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Figure 1. Schematic Diagram of the Complementation Vectors
The pAL5 vector contains the inducible/repressible alcA
promoter, the histone H2A 3'-fragment polyadenylation signal, the
ampicillin resistance gene, and the N. crassa pyr4 gene.
The c15 complementation vector contains the entire c15
ORF, encoding the full-length 332-aa c15 protein, cloned downstream of
the alcA promoter. The pAL5 vector was used as a
negative control for the complementation experiments.
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Complementation studies were performed using the pAL5 vector system.
The entire c15 open reading frame (ORF), encoding the
full-length 332-aa protein, was generated through PCR and was cloned
downstream of the alcA promoter (Fig. 1
). A.
nidulans containing the nudC3 temperature-sensitive
mutation were stably transformed with either the c15 vector or the pAL5
vector. After transformation, 11 c15 vector-transformed strains and 13
pAL5 vector-transformed strains were obtained and assayed for
complementation of the nudC3 mutation at the restrictive
temperature (42 C), on the appropriate media (glycerol).
c15 Complements nudC3 Mutants
The A. nidulans nudC3 mutant strain is temperature
sensitive (Fig. 2A
). At the permissive temperature, 32
C, the colonies are similar to the wild type strain as they are large,
white, have undergone normal growth and differentiation, and are able
to generate spores. However, at the restrictive temperature, 42 C, the
nudC3 colonies are much smaller than the wild type colonies
grown at the restrictive temperature, brown in color, restricted in
cellular growth, and unable to undergo normal differentiation to form
conidia.

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Figure 2. c15 Complements nudC3 Mutants
A, Temperature-sensitive phenotype of the nudC3 mutants.
Wild type A. nidulans and A. nidulans
carrying the nudC3 temperature-sensitive mutation were
plated on MAG + UU at either 32 C or 42 C. The left
panel shows the normal colony growth at 32 C, while the
right panel shows the restricted overall development of
nudC3 mutants at 42 C as compared with the wild type
strain. Bar, approximately 10 mm. B, Phenotype of the
transformed nudC3 mutants. Two independently isolated
A. nidulans strains carrying either the c15 or pAL5
construct were plated on MAG (top) and glycerol minimal
media plates (GLY, bottom) at 32 C (left)
and 42 C (right) for 2 days. Only those strains carrying
the c15 construct grew on the glycerol minimal media plates at 42 C.
Bar, approximately 10 mm.
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After transformation of the nudC3 mutant strain, the
transformants were tested for their ability to complement the
temperature-sensitive nudC3 mutation. All 11 c15
vector-transformed strains complemented the nudC3 mutation
at the restrictive temperature on glycerol media. In contrast, all 13
pAL5 vector-transformed strains failed to complement the
nudC3 mutation at 42 C on glycerol media. To illustrate the
complementation phenotype, two representative transformants of each of
the two constructs, c15 (designated c15#1 and
c15#2) and pAL5 (designated pAL5#1 and
pAL5#2), were grown on either MAG or glycerol minimal media
at the permissive or restrictive temperatures (Fig. 2B
). A.
nidulans containing either construct grew well on either of the
two different media at 32 C. However, when the transformants were grown
at the restrictive temperature, only those on the glycerol minimal
media containing the c15 construct grew. The c15 transformants grew in
a similar manner on glycerol minimal media at either 32 C or 42 C.
Therefore, the results of this experiment indicate that the rat c15
protein has the ability to complement the functions lost by the mutant
A. nidulans NUDC protein and restore normal growth and
differentiation.
c15 Protein Is Expressed in Complemented A.
nidulans
To verify that the c15 protein was being expressed in the
c15-transformed and -complemented A. nidulans, Western blot
analysis of the total cellular proteins was performed using
affinity-purified rabbit anti-c15-carboxy-peptide (c15-C) antibodies
(S. M. Morris, in preparation). The expression of the 45-kDa c15
protein (arrow) was detected in the two c15-complemented
strains tested (Fig. 3
, lanes 2 and 3) and not in the
control nudC3 strain (Fig. 3
, lane 1) or those strains
containing the pAL5 vector (Fig. 3
, lanes 4 and 5). Additionally, the
level of c15 protein expression in the two different c15-transformed
strains varied. The c15#1 strain (Fig. 3
, lane 2)
consistently expressed more c15 protein than did the c15#2
strain (Fig. 3
, lane 3). Furthermore, the c15-C antibodies specifically
recognized a 22-kDa protein band (asterisk), corresponding
to the mutant NUDC protein in all protein preparations. Interestingly,
the overall levels of endogenous mutant NUDC in the c15-expressing
strains was lower than the levels of mutant NUDC found in the
nontransformed nudC3 strain or in the two pAL5-transformed
strains. Both the 45-kDa and 22-kDa protein bands could be specifically
competed by the addition of c15-C to the immunoblotting solutions (data
not shown).

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Figure 3. Expression of c15 Protein in Complemented A.
nidulans
Total cellular proteins (50 µg per lane) from two c15-transformed
strains (c15#1, c15#2) (lanes 2 and 3), two
pAL5-transformed strains (pAL5#1, pAL5#2)
(lanes 4 and 5), and the parental nudC3 mutant strain
(lane 1) were resolved by 12% SDS-PAGE and transferred to a nylon
filter. Primary antibodies, affinity-purified rabbit anti-c15-C, were
applied in a 1:500 dilution for 3 h. Secondary antibodies, donkey
anti-rabbit IgG conjugated to horseradish peroxidase, were then added
in a 1:2000 dilution for 1 h. The arrow indicates
the 45-kDa c15 protein band, and the asterisk indicates
the 22-kDa mutant NUDC protein band.
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Nuclear Movement Phenotype of Complemented A.
nidulans
To better characterize the complemented phenotype, the nuclei of
the transformed A. nidulans were stained with
4',6-diamidino-2-phenylindole (DAPI) to determine whether or not normal
nuclear migration was occurring. A. nidulans were grown for
18 h on coverslips in minimal media containing glycerol at either
32 C or 42 C. When A. nidulans containing the
nudC3 mutation were grown at 32 C, their nuclei migrated
normally into the germ tube and were maintained at equal distances from
each other (Fig. 4A
). In contrast, the nuclei of
A. nidulans containing the nudC3 mutation, when
grown at 42 C, divided but failed to migrate out into the germ tube
(Fig. 4C
). When nudC3 mutant A. nidulans were
transformed with the c15 complementation construct and were grown at 42
C on glycerol minimal media, their phenotype closely resembled that of
the nontransformed nudC3 mutants grown at the permissive
temperature (32 C). The c15-complemented A. nidulans
extended and elongated their germ tube (Fig. 4E
), and their nuclei
migrated into the germ tube of the developing germling.

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Figure 4. Nuclear Movement Is Restored in Complemented
A. nidulans
Spores from the nudC3 mutant strain were germinated and
grown at 32 C and 42 C in glycerol minimal media + UU, and spores from
a c15-transformed strain were grown in glycerol minimal media at 42 C
for 18 h. The germlings were stained with DAPI to allow for
visualization of the nuclei. Panels A, C, and E show the results of the
DAPI staining, while panels B, D, and F show the phase contrast view of
the same field. Bar, approximately 10 µm. Panel G
shows the overall percentage of A. nidulans germlings that
moved their nuclei under the growth conditions described above.
DAPI-stained germlings were scored as "no movement" if none of
their nuclei entered the germ tube or as "movement" if one or more
of their nuclei entered the germ tube. N represents the number of
germlings scored for each growth condition.
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To further quantitate the percentage of A. nidulans that
moved their nuclei under the various growth conditions, DAPI-stained
germlings were counted and scored for their ability to move their
nuclei (Fig. 4G
). In nudC3 mutants, grown at 32 C in
glycerol media, more than 95% of the germlings moved their nuclei. In
contrast, when nudC3 mutants were grown at 42 C in glycerol
media, only approximately 18% of the germlings counted showed any form
of nuclear movement. When the c15-transformed A. nidulans
were grown at 42 C in glycerol media, nearly 90% of the germlings
moved their nuclei. Therefore, c15 is capable of restoring normal
nuclear migration.
c15 Protein Expression Restores NUDF Expression
Endogenous levels of NUDF decrease when nudC3 mutants
are grown at the restrictive temperature of 42 C. Studies involving the
use of an exogenous promoter to induce nudF gene expression
revealed a similar decrease in overall NUDF expression when this
construct was transformed into nudC3 mutants that were grown
at 42 C. These results suggest that NUDC regulates NUDF levels in a
posttranscriptional manner (26). To determine whether or not the
expression of c15 at 42 C in nudC3 mutants could rescue NUDF
expression, Western blot analysis of total cellular proteins was
performed using affinity-purified anti-NUDF antibodies. The expression
of the 49-kDa NUDF protein was detected at normal levels in the wild
type strain (Fig. 5
, lane 1) and in the nudA4
mutant strain (Fig. 5
, lane 2) grown at 42 C. However, the expression
of the NUDF protein was greatly decreased in the nudF7
mutant strain and the nudC3 mutant strain (Fig. 5
, lanes 3
and 4) grown at 42 C. When the c15 protein was expressed at 42 C, in
the two c15-complemented strains tested, the expression of NUDF was
restored (Fig. 5
, lanes 5 and 6). The level to which NUDF protein was
restored correlates well with the level of c15 protein expression
observed in the two c15-transformed strains (Fig. 3
). As a control for
the transformation, two control pAL5 vector-containing strains were
tested and the levels of NUDF were decreased to levels observed in the
nudC3 parental strain (Fig. 5
, lanes 7 and 8). The lower
band is nonspecific (dot). Therefore, the results of this
experiment show that c15 expression can rescue the expression of NUDF
at the restrictive temperature.

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Figure 5. Expression of c15 Protein Restores NUDF Expression
Total cellular proteins (50 µg per lane) from wild type (lane 1),
nudA4 (lane 2), nudF7 (lane 3),
nudC3 (lane 4), c15#1 (lane 5),
c15#2 (lane 6), pAL5#1 (lane 7), and
pAL5#2 (lane 8) strains were resolved by 12% SDS-PAGE and
transferred to nitrocellulose. Primary antibodies, affinity-purified
rabbit anti-NUDF, were applied 1:100 for 1.5 h. Secondary
antibodies, goat anti-rabbit IgG conjugated to alkaline phosphatase,
were then added in a 1:2000 dilution for 40 min. The
arrow indicates the 49-kDa NUDF protein. The
dot indicates a nonspecific protein band.
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DISCUSSION
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The c15 gene was originally cloned as a
PRL-responsive gene from rat Nb2 T cells (29). GenBank searches
revealed that the c15 protein exhibits high similarity over its
carboxy-terminal 94 aa to the A. nidulans nuclear movement
protein encoded by the nudC gene. Based on the similarity
that exists between c15 and NUDC, complementation studies were
performed to determine whether c15 could functionally rescue
temperature-sensitive A. nidulans nudC3 mutants. The results
of our experiments show that the expression of the 45-kDa c15 protein
in an A. nidulans nudC3 mutant strain can complement the
temperature-sensitive growth and nuclear movement phenotypes, as well
as restore depleted endogenous NUDF protein to near wild type levels.
These results indicate that c15 functions like NUDC in A.
nidulans and suggest that c15 may play a functional role in Nb2 T
cells similar to the role played by NUDC in A. nidulans. It
is interesting to note that the levels of endogenous NUDC in the
c15-complemented strains are lower than the levels seen in the
nudC3 strain or vector control, pAL5-transformed strains.
Potentially, the expression of the transformed c15 protein may have the
ability to feedback and down-regulate the expression of the endogenous
mutant NUDC protein or in some way affect the stability of the mutant
NUDC protein, thereby allowing the cell to maintain a
"critical" overall level of NUDC/c15 expression.
The nudC3 allele of nudC, like other
temperature-sensitive nud mutants (nudA,
nudF, and nudG) grows very slowly at the
restrictive temperature (12, 25, 26). The nuclei of these various
nud mutants divide normally; however, nuclear migration
through the germ tube is arrested. Biochemical studies have shown that
the nudC3 mutation affects nuclear migration and growth rate
by causing a decrease in the intracellular levels of NUDF, a protein
that is essential for nuclear migration in A. nidulans (26).
Mutations in the nudF gene that cause a reduction in NUDF
protein level also inhibit nuclear movement. Previous work has shown
that the effect of the nudC3 mutation on NUDF levels is
posttranscriptional, and that NUDC and NUDF are not in complex with
each other (26). Recently, a mutation in the A. nidulans
cytoplasmic dynein heavy chain that acts as a bypass suppressor of the
nudF deletion has been discovered (D. A. Willins and N. R.
Morris, unpublished data). This finding suggests that NUDF may serve as
an upstream regulator of dynein/dynactin function. In A.
nidulans, cytoplasmic dynein is the main motor for nuclear
migration, and evidence derived from characterization of the
nudC3 mutant phenotype indicates that the nudC
gene product probably acts as an upstream modulator of dynein function.
Interestingly, the Drosophila nudC gene shows a genetic
interaction with the Drosophila Glued gene, which encodes a
component of dynactin (J. Cunniff, R. Warrior, Y. Chiu, and N. R.
Morris, unpublished data). As a result of these genetic studies, a
hierarchy can be established in which NUDC can be placed upstream of
NUDF, and both NUDC and NUDF can be placed upstream of dynein and
dynactin. Whether or not the NUDC protein directly affects cytoplasmic
dynein or its functional partner, dynactin, is not known (19, 34).
The nudF gene encodes a 49-kDa WD-40 protein with a
putative coiled-coil domain, similar to the ß-subunit of
heterotrimeric G proteins (26, 35, 36). However, NUDF most closely
resembles (42% identity) the human lissencephaly-1 gene
(LIS-1) (27). Lissencephaly is a neuronal migration disease
characterized by the inability of neurons to migrate to their proper
positions in the cerebral cortex (37). There is evidence to suggest
that neurons may migrate by first extending a long process through
which the nucleus and associated organelles then move to establish a
new position for the cell body (38, 39). These observations potentially
link the genes and proteins involved in centrosomal positioning and
nuclear migration with those involved in neuronal migration and brain
development. LIS-1 is 99% identical to the 45-kDa regulatory subunit
of bovine brain platelet-activating factor acetylhydrolase (PAFAH)
(40). The PAFAH enzyme inactivates platelet-activating factor, a lipid
second messenger, by removing the acetyl group at the sn-2
position (41, 42). The structural similarities that exist between
PAFAH, LIS-1, and NUDF suggest that these proteins may be involved in
PAFAH-associated functions. Thus, NUDC may play a role in regulating
the activity or the targeting of the PAFAH complex in mammalian
cells.
Centrosome orientation in T cells appears to be mediated by a force on
centrosomal MTs just as nuclear movement is mediated by a force on SPB
MTs (8, 11). We propose that c15 may play a role in the centrosome
reorientation that occurs when a T cell meets an antigen-presenting
cell or antigen-bearing target cell (3, 43, 44). In this way, the Golgi
apparatus and its associated secretory vesicles are reoriented,
allowing transport of vesicles along the centrosomal MTs toward the
site of contact (3, 44). Although maximal c15 gene induction
in T cells by PRL occurs at the G1/S transition (29), a constitutive
level of c15 protein already exists (data not shown), which may
participate in the rapid centrosome MTOC reorganization process. A
similar kinetics of c15 induction was observed in
IL-3-stimulated premyeloid cells which suggests that c15
induction by cytokines is part of an activation response (29).
Furthermore, the c15 gene product could be involved in
other cytoplasmic dynein-mediated functions other than moving
centrosome and nuclei, e.g. vesicle migration or mitosis
(45, 46), which occur later in the cell cycle as suggested by the
kinetics of c15 induction in PRL-stimulated T cells (29).
This additional functional complexity is also suggested by the large
difference in size between the 45-kDa c15 protein and the 22-kDa NUDC
protein. The size difference is due to a much larger amino-terminal
domain in the rat c15 protein, which contains basic and acidic motifs
that may be involved in protein-protein interactions.
In summary, we have identified a mammalian (rat) gene from
PRL-stimulated T cells, c15, that when expressed in A.
nidulans nudC3 mutants can functionally rescue the nuclear
movement defect. The fact that the rat c15 protein can replace the
nuclear movement function of the A. nidulans nudC gene
suggests that c15 and NUDC have similar functions. Because of the
structural and functional similarities between c15 and NUDC,
c15 is likely a mammalian homolog of nudC, and we
have therefore named it RnudC. Additionally, c15/RNUDC is
widely represented throughout eukaryotes, as immunoreactive
c15/RNUDC-like proteins have been detected in Drosophila (J.
Cunniff and R. Warrior, personal communication), monkey, and man (data
not shown).
Future studies will address the localization of c15/RNUDC in the cell
and its association with other proteins. We are particularly interested
in determining whether c15/RNUDC localizes to the centrosome MTOC or to
the cortex in PRL-stimulated Nb2 T cells. These studies will help
determine the role of c15/RNUDC in the events leading to T cell
activation, proliferation, and differentiation.
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MATERIALS AND METHODS
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A. nidulans Strains and Growth Media
The A. nidulans strains used were AO1 (nudC3;
pabaA1; wA2; nicA2; pyrG89), XX21 (nudF7; pyrG89; yA2),
XX8 (nudA4; pyrG89; wA2; chaA1), GR5 (pyrG89; wA3;
pyroA4), and SJ002 (pyrG89) (12, 25, 26). The
temperature-sensitive nudC3, nudF7, and nudA4
mutants grow normally at the permissive temperature of 32 C, but are
severely restricted in growth at the restrictive temperature of 42 C.
Nontransformed A. nidulans colonies were grown on Malt
Extract Medium (MAG) [2% malt extract (Difco, Detroit, MI), 0.2%
peptone, 1% dextrose, trace elements (1000x solution: 40 mg/liter
Na2B4O7:10H20, 400
mg/liter CuSO4:5H2O, 800 mg/liter ferric
citrate or chloride, 800 mg/liter MnSO4:H2O,
800 mg/liter NaMoO4:2H2O, 8 g/liter
ZnSO4:7H2O), vitamins (500x solution: 0.1%
p-aminobenzoic acid, 0.1% niacin, 0.1% pyridoxine HCl,
0.1% riboflavin, 0.1% thiamine HCl, 0.1% choline HCl, 0.2%
d-biotin), 2% agar] or Yeast Extract Medium (YAG) (0.5% yeast
extract, 1% dextrose, 10 mM MgSO4, trace
elements, vitamins, 1.5% agar) supplemented with 0.12% uridine and
0.11% uracil (UU) (United States Biochemical Corporation, Cleveland,
OH) (24, 47). YAG without agar + UU was used for the growth of liquid
cultures for transformations. After transformation, A.
nidulans colonies were maintained on MAG or YAG plates lacking UU.
For complementation experiments, transformed A. nidulans
were additionally grown on supplemented minimal media plates [2% 50x
salts (50x salts: 300 g/liter NaNO3, 26 g/liter KCl, 24.65
g/liter MgSO4:7H2O), 12 mM
KPO4 pH 6.8, trace elements, vitamins, 1.25% agar]
containing 10 ml/liter glycerol (32). For protein isolation
experiments, A. nidulans were grown in liquid minimal media
containing 10 ml/liter glycerol with or without UU.
Complementation Vectors
The 100A plasmid, containing the entire rat c15 ORF
(29), was used as a template to generate a 1.0-kb DNA fragment by PCR
using Pfu polymerase (Statagene, La Jolla, CA). The upstream
primer contained a KpnI linker (lowercase):
5'-gcgggtaccgATGGGAGGGGAGCAG-3' (start codon
underlined), and the downstream primer contained an
XbaI linker (lowercase):
5'-gcgtctagaCTAGTTGAATTTGGC-3' (stop codon
underlined). The KpnI/XbaI-digested
PCR product, encoding c15, was cloned downstream of the alcA
promoter in the pAL5 vector (32) and confirmed by sequencing. The c15
vector and pAL5 vector were used to transform A. nidulans
containing the nudC3 mutation.
A. nidulans Transformation
Conidia (1 x 109) were inoculated into 50 ml
YAG without agar supplemented with UU. The conidia were allowed to
germinate for about 5.5 h, or until the emerging germ tube was
visible, at 32 C with shaking. The germinated conidia were harvested by
centrifugation, resuspended in 40 ml lytic mix [20 ml Solution A (0.1
M citric acid, 0.8 M
(NH4)2SO4 pH 5.8 with KOH pellets),
20 ml Solution B (1% yeast extract, 2% sucrose, 40 mM
glucose, trace elements, vitamins), 10 mM
MgSO4, 200 mg BSA (Sigma, St. Louis, MO), 100 mg Novozyme
234 (Sigma), 125 µl glucuronidase (Sigma)], and the cell wall was
digested for 2 h at 32 C with shaking. The protoplasts were washed
two times in Solution C (50 mM citric acid, pH 6.0, 0.4
M (NH4)2SO4, 1%
sucrose) and were resuspended in 1 ml Solution E (0.6 M
KCl, 100 mM CaCl2, 10 mM Tris-HCl,
pH 7.5). Protoplasts (100 µl) were added to 6 µg plasmid DNA,
followed by the addition of 50 µl Solution D (25% polyethylene
glycol 8000, 100 mM CaCl2, 0.6 M
KCl, 10 mM Tris-HCl, pH 7.5), and incubated on ice for 20
min. Next, 1 ml Solution D was added to the protoplasts and allowed to
incubate at room temperature for 30 min. Aliquots (200 µl) of the
protoplast mixture were plated in 3 ml 45 C sucrose top agar (0.5%
yeast extract, 20 mM glucose, 1 M sucrose,
trace elements, vitamins, 1% agar) onto sucrose plates (0.5% yeast
extract, 20 mM glucose, 0.2 M sucrose, trace
elements, vitamins, 1.5% agar), and incubated at 32 C for 34
days.
Complementation of nudC3 Mutants with c15
To test for complementation, conidia containing either the c15
or pAL5 construct were streaked on either MAG or glycerol minimal media
lacking UU at 32 C or 42 C. Independently isolated transformants of
each of the two constructs (c15 and pAL5) were plated, and colonies
were allowed to grow for 2 days before analysis. Conidia from the
transformants were isolated and retested for complementation five
times.
Protein Preparation and Western Blot Analysis
To prepare total cellular proteins, 5 x 108
conidia were inoculated into 50 ml supplemented minimal media
containing 10 ml/liter glycerol and incubated for 42 h at 42 C
with shaking. The mycelia were harvested by centrifugation, washed in
ice-cold H2O, collected by filtration through cheesecloth,
and pressed dry. The mycelia were ground in a Tenbroek homogenizer
(Fisher Scientific, Pittsburgh, PA) in 12 ml extraction buffer [50
mM Tris-HCl, pH 7.4, 200 mM NaCl, 5
mM EDTA, 5 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml each of soybean trypsin
inhibitor, aprotinin, leupeptin,
N-tosyl-L-phenylalanine chloromethyl ketone
(Sigma)], and insoluble proteins were removed by centrifugation at
14,000 rpm for 5 min at 4 C. Protein concentration was determined by
Bradford assay (Bio-Rad, Richmond, CA) using BSA as a standard. The
cellular proteins were stored at -20 C.
For Western blotting, 50 µg total cellular proteins were analyzed by
12% SDS-PAGE and transferred to Immobilon P (Millipore, Bedford, MA)
or nitrocellulose (Bio-Rad) as previously described (29). To determine
c15 expression, the blot was blocked with 5% nonfat milk, 0.2% Tween
20 (Sigma) in Tris-buffered saline, and affinity-purified rabbit
anti-c15-C antibodies (S. M. Morris, in preparation) were applied in a
1:500 dilution, followed by the addition of donkey anti-rabbit IgG
antibodies conjugated to horseradish peroxidase (Amersham, Arlington
Heights, IL) in a 1:2000 dilution. c15 proteins were detected with the
enhanced chemiluminescence system as suggested by the manufacturer
(Amersham). To detect NUDF, affinity-purified rabbit anti-NUDF
antibodies were used at a 1:100 dilution, followed by goat anti-rabbit
IgG antibodies coupled to alkaline phosphatase in a 1:2000 dilution,
and developed with 5-bromo-4-chloro-3-indoyl phospate
p-toluidine salt and p-nitro blue tetrazolium
chloride (26).
DAPI Staining of Nuclei
To stain the nuclei of the developing germlings, 5 x
106 conidia were inoculated into Petri dishes containing
sterile coverslips and 25 ml supplemented liquid minimal media with 10
ml/liter glycerol and were incubated at either 32 C or 42 C for 18
h. The coverslips with the attached germlings were rinsed in
H2O and placed in methanol at -20 C for 10 min, rinsed
well with H2O, and placed in acetone at -20 C for 10 min.
The coverslips were rinsed again and placed in a 50 ng/ml DAPI (Sigma)
solution for 10 min. After a final rinse, the coverslips were mounted
in ProLong Antifade (Molecular Probes, Inc., Eugene, OR) and viewed at
1000x using the Zeiss Axiophot system (Zeiss, Jena, Germany) (Baylor
Integrated Microscopy Core, Baylor College of Medicine, Houston,
TX).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Stephen A. Osmani for the A. nidulans
nudC3 strain and Dr. Sophia Tsai for her critical comments on this
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Li-yuan Yu-Lee, Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.
This work was supported by grants from the American Cancer Society
(BE-49J) (to L.-y. Y.-L.) and The Linda and Ronald Finger Lupus
Research Center (to S. M. M.)
Received for publication September 9, 1996.
Revision received November 19, 1996.
Accepted for publication November 21, 1996.
 |
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