First published online 25 February 2003
doi: 10.1242/jcs.00337
Chlamydomonas DIP13 and human NA14: a new class of proteins associated with microtubule structures is involved in cell division
Frank Pfannenschmid1,
Verena C. Wimmer1,
Rosa-Maria Rios2,
Stefan Geimer3,
Ulla Kröckel1,
Andreas Leiherer1,
Katrin Haller1,
Yvonne Nemcová4 and
Wolfgang Mages1,*
1 Lehrstuhl für Genetik, Universität Regensburg, 93040 Regensburg,
Germany
2 Departamento de Microbiologia, Facultad de Biologia, Universidad de Sevilla
Apdo. 1095, 41080-Sevilla, Spain
3 Botanisches Institut, Universität zu Köln, 50931 Köln,
Germany
4 Laboratory of Gene Expression, 1st Faculty of Medicine, Charles University and
Department of Cell Biology, Institute of Experimental Medicine, Academy of
Sciences of the Czech Republic, Prague, Czech Republic
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Fig. 7. DIP13 antisense RNA experiments. (A) pVW1 antisense construct. The
AR-promoter (AR-P), DIP13 inverse reading frame (DIP13-ORF)
and the RBCS2 gene 3' sequences (RBCS2-3') are indicated.
Single restriction sites XbaI, BamHI, and KpnI are
given for orientation. Primers (P1, P2) for amplification of a specific 560-bp
fragment (open box) are indicated at their respective positions. (B) Result of
PCR analysis with primers P1 and P2 and genomic DNA of three putative
transformants (#33, 45 and 67) and control genomic DNA from the untransformed
parent strain (wt). Controls were done without added template (–
control) or 1 ng pVW1-DNA (+ control). St, DNA size standard. For orientation,
some fragment sizes (in bp) are indicated to the right. (C) Phenotypic
comparison of antisense transformants (C1-3) and untransformed cells (C4).
Bar, 10 µm. (D) Analysis of DIP13 protein reduction in antisense strains
#33, 45 and 67. Left panel: Coomassie-stained gel as loading control. Lane A:
extract from the untransformed strain. Middle panel: immunoblot with the same
amounts of protein per lane of the same four strains probed successively with
anti-DIP13 antibody (DIP13), anti- -tubulin antibody (-Tub) and
anti-L23 antibody (L23). Right panel: result of densitometric analysis of
DIP13 protein levels derived from the immunoblots shown. For standardization
of loading, the signals obtained with anti L23 antibody were used. (E)
Indirect immunofluorescence with anti-DIP13 antibody (E1) and DAPI staining
(E3) of a transformed cell of strain #45 showing typical labeling (compare
Fig. 4) at the two basal body
spots (E1, white arrows) and two nuclei (E3, white arrows) at opposite poles.
E2, corresponding phase image. Bar in C4 (10 µm) applies to all microscopic
images.
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Fig. 2. Comparison of the derived DIP13 protein with its human and mouse homologs,
physical map and Southern analysis. (A) Comparison of the derived DIP13
protein with two known homologs, human NA14 and a mouse unnamed protein.
Residues conserved in at least two of three proteins are shown in bold. The
region encoded in plasmid pDIP is underlined. A potential microtubule binding
site is boxed, potential phosphorylation sites for casein kinase II (double
headed arrow) or protein kinase C (open rectangles), a putative
N-glycosylation site (thick black line) and the conserved leucine residues of
the leucine zipper-like N-terminal motif (*) are indicated above the sequence.
(B) DIP13 gene structure consisting of three exons (black boxes) and
two introns (white boxes). 5' and 3' untranslated regions are
shown as grey boxes, positions of translational start (ATG) and stop (TGA)
codons as well as five polyadenylation signals (P) are indicated. (C) Southern
analysis of C. reinhardtii genomic DNA (strain 125 MT+) with a
DIP13 cDNA probe performed under stringent conditions suggests that
DIP13 is a single copy gene. St, molecular size standard; sizes are
indicated in kb; Ps, PstI; Pv, PvuII; St, StuI.
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Fig. 3. Specificity of anti-DIP13 antiserum and DIP13 expression during
the cell cycle. (A) Characterization of a polyclonal anti-DIP13 antiserum
produced in rabbit. (Left panel) Crude cell extracts from 106
C. reinhardtii cells per lane were probed with preimmune serum (P;
1:500) as well as crude (C, 1:500) and affinity-purified (A, 1:100) anti-DIP13
antiserum and detected by enhanced chemiluminescence. Sizes of reacting bands
are indicated to the left. (Right panel) Crude cell extracts of 106
C. reinhardtii cells per lane were probed with affinity-purified
anti-DIP13 antibody preincubated with 80 µg recombinant DIP13 per ml
incubation buffer (A-PI) or affinity-purified anti-DIP13 antibody (A, 1:100).
The size of the reacting band is indicated to the right. (B) The 24 hour
C. reinhardtii life cycle under a synchronizing 16 hour light/8 hour
dark regimen. Every full hour 100 cells from a synchronous culture were
analyzed by light microscopy and absolute numbers of cells in the 1-, 2-, 4-
or 8-cell stage were noted in the table shown on the right. (C) Western
analysis with samples isolated from the same culture as in B during the period
of cell division. Equal amounts of protein per lane were probed with
affinity-purified anti-DIP13 antibody (DIP13), anti--tubulin antibody
(Tub) and anti-L23 antibody (L23). Time points of protein sampling correlate
with time points in B. The same membrane was incubated successively with the
three primary antibodies. Exposure times to film were 1 minute in each case.
(Lower panel) Coomassie-stained gel of identical protein samples as a loading
control. St, standard lane.
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Fig. 5. Immunogold labeling (postembedding technique) of basal bodies and flagella
using anti-DIP13 and anti-rabbit-IgG conjugated to 10 or 15 nm gold particles.
(A1-3) Longitudinal sections of the basal body region with labeling at the
outside of one basal body distal to the neighbouring basal body (A1,2) and
labeling of triplet microtubules inside the basal body (A3). (A4-7)
Cross-sections from distal to proximal with respect to the cell body
demonstrate the presence of DIP13 in several structurally defined zones of the
basal bodies. Gold particles are pointed out by black arrowheads. (B)
Longitudinal and cross-sections of flagella. Both outer doublet (B1-3) and
central pair (B4-6) microtubules are decorated. In some sections (B7-10) no
decision can be made. Labeling does not show any periodicity but as indicated
in B4 and B9 particles are often found in close proximity (black arrowheads).
Bars, 200 nm. Bar shown in A3 applies to all figures except A5.
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Fig. 6. NA14 localization to centrosomes in human cells (A) Left panel: Recombinant
DIP13 (lane 1) and NP-40-soluble fraction from COS-7 cells transfected with a
HA-tagged version of NA14 (lane 2) were analyzed with anti-NA14 antibody
(Probe:anti-NA14;1:100). Positions of molecular weight standards are indicated
at the right. Middle and right panels: results of immunoprecipitation
experiments performed from a solution containing recombinant DIP13 (lanes 3,7)
and from extracts of HA-NA14 overexpressing cells, with 10 µl of anti-DIP13
antibody (lanes 5,9) linked to protein A-Sepharose beads. Negative controls
were done from the same HA-NA14-overexpressing cell extracts with 10 µl of
preimmune serum linked to protein A-Sepharose beads (lanes 4,8). After
washing, bead pellets and supernatants (lanes 6,10) were analyzed by
immunoblotting using anti-NA14 antibody (central panel, lanes 3-6) or anti-HA
antibody (right panel, lanes 7-10). (B) DIP13/NA14 localizes to centrosomes in
HeLa cells and both basal bodies and flagella in human spermatozoa. HeLa cells
or human spermatozoa (bottom panels) were double-stained with anti-DIP13
antibody (left panels, green) and anti- -tubulin, CTR453 or
anti--tubulin (central panels, red). Separate green and red images were
collected and merged (right panels). Yellowish staining indicates
colocalization of both labelings. Arrows indicate centrosomes or basal bodies.
Bars, 10 µm. (C) Triton-soluble and insoluble fractions of KE37 cells and a
preparation of isolated centrosomes were resolved by SDS-PAGE, blotted and
probed with anti-NA14 antibody.
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© The Company of Biologists Ltd 2003
