Department of Biology, Yonsei University, Seoul, Korea 120-749
We have examined the distribution of four
mRNAs-tubulin,
-tubulin, flagellar calmodulin,
and Class I mRNA
during differentiation of Naegleria
gruberi amebas into flagellates by in situ hybridization. Three of the four mRNAs
-tubulin,
-tubulin, and
Class I mRNA
began to be colocalized at the periphery of the cells as soon as transcription of the respective
genes was activated and before any microtubular structures were observable. At 70 min after the initiation of differentiation, these mRNAs were relocalized to the
base of the growing flagella, adjacent to the basal bodies and microtubule organizing center for the cytoskeletal microtubules. Within an additional 15 min, the
mRNAs were translocated to the posterior of the flagellated cells, and by the end of differentiation (120 min), very low levels of the mRNAs were observed.
Cytochalasin D inhibited stage-specific localization of
the mRNAs, demonstrating that RNA localization was
actin dependent. Since cytochalasin D also blocked differentiation, this raises the possibility that actin-dependent RNA movement is an essential process for differentiation.
NAEGLERIA gruberi amebas differentiate into swimming flagellates in less than 2 h after initiation of
differentiation (6). During differentiation, N. gruberi changes its shape sequentially from an amorphous
ameba to a sphere and then to a flagellate with a regular
contour, forming two basal bodies, two flagella, and cytoskeletal microtubules (CSMT)1 de novo (see Fig. 7). Using
a monoclonal antibody against Naegleria
The basal body is the microtubule organizing center for
flagellar (or ciliary) axonemal microtubules and is structurally identical to a centriole. In a few organisms and cell
types, including Naegleria gruberi, the basal body or its
surrounding material seems to act as a microtubule organizing center in the formation of cytoskeletal microtubules
(3, 12, 14). However, the mechanism of basal body formation and its function in the formation of the microtubule systems still remain to be elucidated.
While the composition and assembly of the basal body
are poorly understood, it could be assumed that the local
concentration of the component proteins should be high
enough to initiate the assembly of the organelle and that
the colocalization of the component proteins in a limited
area of the cell would greatly facilitate this process.
Localization of mRNA is one of the possible mechanisms by which a cell can concentrate a protein in a specific area (13, 22, 26). Using in situ hybridization, we examined the distribution of four mRNAs that are transiently
and coordinately accumulated during differentiation of N. gruberi, differentiation-specific (DS) mRNAs (19, 20). Two
of the four DS mRNAs encode Cell Growth and Differentiation
Growth and differentiation of N. gruberi strain NB-1 was carried out as
described elsewhere (6, 19).
Cell Fixation
Differentiating cells were fixed in a formaldehyde fixation buffer (1× fixation buffer; 25 mM sodium phosphate, pH 7.2, 1% formaldehyde, 63 mM
sucrose, 1 mM Tris-HCl [25]). 200 µl of differentiating cells (107 cells/ml)
and 200 µl of 2 mM Tris-HCl, pH 7.6, at 20°C were added into 400 µl of
2× fixation buffer and incubated for 5 min at 4°C. 70 µl of the fixed cells
were applied to a glass slide coated with gelatin-chromium potassium sulfate (27) and air dried for 3-4 h at room temperature. The fixed cells were
treated with 0.1% NP-40 in the fixation buffer for 5 min at room temperature, washed four times with PBS buffer, and then dried at room temperature. The fixed and permeabilized cells were then soaked sequentially in
methanol and acetone for 10 min at 4°C.
Probe Preparation
A cDNA fragment of each mRNA (PstI restriction fragment of pcNg 8-5,
580 bp, for In Situ Hybridization
In situ hybridization with the DIG-labeled cDNA probes was carried out
as suggested by the manufacturer. The cells were prehybridized for 3 h
with 100 µl of prehybridization buffer (50% formamide, 5× SSC, 5× Denhardt's solution, 4 mM EDTA, 100 µg/ml wheat germ tRNA) per slide at
45°C in a moist chamber. The prehybridization buffer was removed carefully with filter paper, and 20 µl of hybridization buffer (the same as prehybridization buffer without the tRNA) with DIG-labeled denatured
cDNA probe (~2 ng/µl) was added to each slide glass. Each slide was covered with a siliconized cover glass and sealed with rubber cement to prevent evaporation of the hybridization buffer. Hybridization was carried
out at 45°C for 6 h in a moist chamber. After hybridization, the slides were washed twice with 3× SSC, twice with 0.3× SSC, and then twice with 0.2×
SSC (15 min for each wash).
Determination of mRNA Location
After hybridization, location of each mRNA was determined by alkaline
phosphatase-conjugated anti-DIG antibody with Nitro blue tetrazolium
and 5-bromo-4-chloro-3-indolyl-phosphate as substrates using a kit from
Boehringer Mannheim.
Propidium Iodide Staining
Nuclei were visualized by propidium iodide staining of the cells. After in
situ hybridization with DIG-labeled cDNA probes and immunological detection of respective mRNAs, the samples were stained with propidium
iodide (500 µg/ml in 50% glycerol, 0.5 M Tris-HCl, pH 7.5, 2% 1,4-diazabicyclo-[2,2,2]-octane) for 10 min. The stained cells were washed three
times with PBS and examined by fluorescence microscopy with a
rhodamine filter.
Localization of the Tubulin mRNAs
Cells were taken at 0, 20, 40, 70, 85, and 120 min after initiation of the differentiation. The cells were fixed and hybridized with DIG-labeled cDNA probes, and the distribution
of the DS mRNAs was determined by anti-DIG antibody
conjugated with alkaline phosphatase as described in Materials and Methods.
When a probe for
Table I.
Localization of the DS mRNAs
-tubulin, Walsh
showed that Naegleria amebas do not have microtubulebased structures except for the mitotic spindle fibers in dividing cells (25). Microtubule structures first appear in the
cytoplasm of a cell ~50-55 min after initiation of differentiation, and then two basal bodies are formed on the cell
periphery. Two flagella begin to appear from the basal bodies, and, at 70 min after initiation of differentiation, 50% of
the cells have visible flagella. When the flagella reach about
10 µm in length, 80 min after initiation, a complex array of
CSMT is observed radiating from the base of the flagella
at which the basal bodies are located. At 120 min after the
initiation, the flagella reach full length (~15 µm) and the
cytoskeletal microtubules elongate, reaching the distal end
of the cell (25). Thus, the formation of basal bodies appears to be the key step in the formation of the flagella and
the CSMT during the differentiation of N. gruberi.
Fig. 7.
A schematic presentation of localization of the DS mRNAs during differentiation of N. gruberi amebas into flagellates. This diagram is drawn based on data of our own and from references 6, 10, and 25. Formation of CSMT from the base of flagella is not
shown. The dotted area represents the site of localization of the DS mRNAs.
[View Larger Version of this Image (15K GIF file)]
- and
-tubulin, the major
components of flagellar axoneme and cytoskeletal microtubule system (21). The third DS mRNA, the flagellar calmodulin mRNA, encodes a calmodulin (21) that is found
in the flagellar axoneme but not in the cell body (7, 10).
The protein product of the fourth DS mRNA that is ~7-kb
long has not been identified (Class I mRNA [20]). We report here that these DS mRNAs are specifically localized
in a specific area from which the flagella grow, and we
present evidence that this specific localization of the DS mRNAs could have an important role in the formation of
the microtubule systems during the differentiation of Naegleria amebas into flagellates.
Materials and Methods
-tubulin [20]; EcoRI fragment of
13, 450 bp, for
-tubulin
[4]; PstI fragment of pcNg 1-8, 520 bp, for Class I [20]; PstI fragment of
pcNg 44, 375 bp, for flagellar calmodulin [20], and PstI fragment of pcNg
3-28, 700 bp, for a nonspecific mRNA [20]) was labeled with digoxygenin
(DIG)-11-dUTP by using a DIG DNA labeling and detection kit (Boehringer Mannheim, Mannheim, Germany).
Results
-tubulin mRNA was used, no
-tubulin
mRNA was detected in amebas (0 min, Fig. 1 A) as expected from the previous reports (19, 20). At 20 min after
the initiation,
-tubulin mRNA is actively transcribed
(19), and the amount of this mRNA has reached about 20 to 30% of the peak value (20). At this stage, the
-tubulin
mRNA was detected in 76% of cells, and in 74% (56 out
of 76) of the labeled cells, the
-tubulin mRNA was concentrated at one location (Fig. 1 B; Table I). In the rest of
the labeled cells, the tubulin mRNA was concentrated at
two, or rarely three, close locations (Fig. 1, B and C; Table I; also see Fig. 1 I for
-tubulin mRNA).
Fig. 1.
Localization of DS mRNAs during the differentiation of N. gruberi. Differentiating cells were taken at various stages of differentiation and fixed as described in Materials and Methods. The fixed cells were used for in situ hybridization with cDNA probes labeled
with DIG. Location of each mRNA probe was determined by using anti-DIG antibody conjugated with alkaline phosphatase. (A-H)
Location of -tubulin mRNA. (A) 0 min; (B) 20 min; (C) 40 min; (D and E) the same cells at 70 min at different focal planes to show the flagella; (F and G) the same cells at 85 min at different focal planes to show the flagella; (H) 120 min. (I-L) Location of
-tubulin mRNA; (I) 40 min; (J and K) the same cells at 70 min at different focal planes to show the flagella; (L) 85 min. (M-P) Location of Class
I mRNA. (M) 40 min; (N and O) the same cells at 70 min at different focal planes; (P) 85 min. (Q-T) Location of flagellar calmodulin
mRNA. (Q) 40 min; (R and S) the same cells at 70 min at different focal planes; (T) 85 min. Black arrowheads indicate locations of the DS
mRNAs, and open arrowheads point to the flagella. The pictures were taken using a microscope (model Optiphot-2; Nikon, Inc.,
Melville, NY) under differential interference contrast (DIC) conditions. Bar, 10 µm.
[View Larger Version of this Image (108K GIF file)]
At 40 min after the initiation, most of the cells were still
ameboid in shape (6). At this stage, transcription of the -tubulin mRNA is most active (19), and the amount of
the mRNA has reached about 60% of its peak value (20).
At this stage, the
-tubulin mRNA was detected in 81% of
cells, and in 82% of the cells (66 out of 81), the tubulin
mRNA was localized in one area. In most of these cells,
the
-tubulin mRNA was located at the periphery (Fig. 1
C; Table I).
At 70 min, most of the cells have rounded up into
spheres, about 50% of the cells have visible flagella on the
surface (6, 25), and the amount of the tubulin mRNA is at
its peak (20). At this stage, strong -tubulin mRNA-specific staining was observed in 84% of cells (Fig. 1, D and
E). In the majority of these cells (91%, 76 out of 84; Table
I), the
-tubulin mRNA was concentrated in one area.
This specific localization was more evident in the flagellated cells. The
-tubulin mRNA was localized in one area in 94% of the flagellated cells. When we examined the location of the tubulin mRNA in flagellated cells with one
labeled spot, this
-tubulin mRNA-specific staining was
located at the base of the growing flagella in 82% of the
flagellated cells (Table II).
Table II. Translocation of the DS mRNAs in Flagellated Cells |
At 85 min after initiation, most of the cells have visible
flagella, the amount of tubulin mRNA has decreased to
80% of the maximum (20), complex array of the CSMT is
rapidly growing (25), and most of the flagellated cells have
already started to elongate (6). At this stage, the -tubulin
mRNA was still found to be concentrated in a specific area
in 90% of the cells (Table I). However, the
-tubulin
mRNA was not found at the base of the flagella in most of
the flagellated cells. Instead, the tubulin mRNA was found
at the posterior or in the middle of the flagellated cells (Fig. 1, F and G; Table II).
To see this translocation of the mRNA from the base of flagella to the posterior of the flagellates and the relationship between the translocation and the change in the cell shape, we quantitated the number of flagellates having one labeled spot based on their shape and on the location of the mRNA in the cells at 70 and 85 min (Table II).
At 70 min, 93% of the flagellated cells were round in
shape. In 88% (82 out of 93) of these round flagellates, the
-tubulin mRNA was found at the base of the flagella. In
the rest of the round flagellates, the
-tubulin mRNA was
located mostly in the middle of the cells (Table II). In the
elongated cells (7%), the
-tubulin mRNA was not found
at the base of the flagella but in the middle or at the posterior of the cells (Table II). At 85 min, the majority of flagellated cells (78%) had started to elongate, and 22% of
flagellated cells were still round in shape. In the elongated
flagellates, the
-tubulin mRNA was found in the middle (45%, 35 out of 78) or at the posterior (55%, 43 out of 78)
of the cells. In the round flagellates, the tubulin mRNA
was located at the base of the flagella or in the middle of
the cells (Table II). By 120 min, the differentiation is completed and the concentration of the mRNA has fallen below 10-20% of the maximum value (20). At this stage, the
-tubulin mRNA was still concentrated at a specific area
in about half of the cells (in most cases, at the posterior)
but the staining was faint (Fig. 1 H; Table I).
These results show that the -tubulin mRNA is concentrated at a specific area of a differentiating cell, that at 70 min this mRNA is localized at the base of the growing flagella, and that the mRNA is translocated from the base of
the flagella to the posterior as the cells differentiate. These
results also suggest that the translocation of the
-tubulin
mRNA begins before the initiation of cell elongation.
The distribution of -tubulin mRNA during N. gruberi
differentiation was essentially identical to that of the
-tubulin mRNA (Fig. 1, I-L; Tables I and II) except at 120 min.
Even though the stainings were faint like those of the
-tubulin mRNA, we observed the
-tubulin mRNA in 82% of
cells at this stage, and the mRNA was concentrated at two
or more regions in 70% of the stained cells (20). The distribution of the Class I mRNA, a DS mRNA of unknown
protein, was similar to that of the
- and
-tubulin mRNA.
In amebas, the Class I mRNA was not detected (data not
shown). However, after 40 min of differentiation, the Class I mRNA was found concentrated at the periphery of the
cells (Fig. 1 M). In the later stages of differentiation, the
Class I mRNA was distributed in the same way as the
- and
-tubulin mRNAs (Fig. 1, N-P).
Localization of the Flagellar Calmodulin mRNA
The distribution of the flagellar calmodulin mRNA that encodes a flagellar-specific calmodulin (7) was very similar to those of the other three mRNAs in early stages (up to 40 min), but it was quite different at later stages (at 70 and 85 min). This mRNA was not detected in amebas like the other three mRNAs (data not shown). At 40 min, at which time transcription of this gene is most active (19), the mRNA was detected in 78% of the cells (Table I). In 63% (49 out of 78) of these cells, the flagellar calmodulin mRNA was found in one area, and in most of the cells, the mRNA was located at the cell periphery (Fig. 1 Q; Table I).
At 70 min, the flagellar calmodulin mRNA was detected in 85% of cells, and in 83% (70 out of 85) of these cells, the mRNA was localized at one area (Table I). However, location of the mRNA relative to that of the flagella and to the shape of cells was quite different from those of the other three DS mRNAs. The flagellar calmodulin mRNA was localized at the base of flagella in only 16% of round flagellates. In 77% of the round flagellates (73 out of 95), the mRNA was located in the middle of the cells (Fig. 1, R-S; Table II).
At 85 min, the flagellar calmodulin mRNA was still specifically localized in 80% of cells, and the mRNA was localized at one area in 90% of the cells (72 out of 80) (Fig. 1 T; Table I). In most round flagellates of this stage, the flagellar calmodulin mRNA was located in the middle of the cells (16 out of 18). In elongated flagellates, the mRNA was found at the posterior (67%) or in the middle of the cells (33%) (Table II). At 120 min, only a faint staining was observed (data not shown).
These results show that the flagellar calmodulin mRNA is also specifically localized and that the location of the flagellar calmodulin mRNA changes during the differentiation. These results also show that translocation of the flagellar calmodulin mRNA begins before the onset of translocation of the other three DS mRNAs.
Colocalization of the DS mRNAs during the Differentiation
The distribution of the three DS mRNAs (-tubulin mRNA,
-tubulin mRNA, and Class I mRNA) at 70 and 85 min
suggested that they are colocalized during the differentiation. To test this possibility, we performed two sets of in
situ hybridization experiments. In one set of the experiments, locations of
-tubulin mRNA and
-tubulin mRNA
were examined simultaneously by adding the respective DIG-labeled cDNA probes in one hybridization reaction.
If the two mRNAs were not colocalized, we would expect
several (at least two) distinctly stained regions. As shown
in Fig. 2, A (20 min) and B (40 min), the staining pattern
was similar to that of Fig. 1, B and C, where only the
-tubulin cDNA probe was used. These results were summarized
in Table III. We observed one stained region in 58% of
stained cells at 20 min, 74% of the cells at 40 min, and 91%
of the flagellated cells at 70 min. When the locations of
tubulin mRNA and Class I mRNA were examined in the
same way, we obtained similar results (Fig. 2, C and D).
These data suggest that the three DS mRNAs are colocalized during differentiation.
Table III. Colocalization of the DS mRNAs |
Because the distribution of the flagellar calmodulin
mRNA was similar to that of the tubulin mRNAs at early
stages, we examined whether the flagellar calmodulin
mRNA was also colocalized with the other three mRNAs
at early stages. In these experiments, we examined the location of the flagellar calmodulin mRNA and that of the
-tubulin mRNA simultaneously in 20-, 40-, and 70-min
cells. We observed one stained region in 48% of the
stained cells at 20 min and 73% of the stained cells at 40 min (Table III and Fig. 2 E). However, in flagellated cells
at 70 min, we observed only 52% of the cells having one
stained region. In the rest of the flagellated cells (48%),
we observed two or more well-separated stained regions
(two spots, 38%; three or more, 10%; Fig. 2 F). These results suggest that the flagellar calmodulin mRNA is colocalized with the other three DS mRNAs at early stages of
differentiation. These results are also consistent with the
observed fact that the flagellar calmodulin mRNA was
found at the base of the flagella only in 16% of the flagellated cells at this stage (Table II) and support our rationale behind the method that we used to show the colocalization of the DS mRNAs.
The Localization Is Specific to the DS mRNAs
This localization of the DS mRNAs could be the result of
active transcription of the DS genes in the nucleus, especially at early stages of differentiation when the genes are
actively transcribed (19). To test this hypothesis, we
stained the 40-min cells, in which the DS genes are being
most actively transcribed, with propidium iodide after in
situ hybridization with the DS cDNA probes. As shown in
Fig. 3, A and B, the location of -tubulin mRNA was
clearly cytoplasmic, distinct from that of the nucleus. This
result showed that the specific localization was not the result of active transcription and of concentration of the
mRNAs in the nucleus.
The specific localization of the DS mRNAs was not a
general phenomenon of N. gruberi differentiation. We examined distribution of another Naegleria mRNA that is
present at high concentrations both in amebas and in flagellates, a nonspecific mRNA (19, 20). Unlike the DS mRNAs,
the nonspecific mRNA showed uniform distribution throughout the differentiation (Fig. 4, A and B; and data not shown). The specific localization of the DS mRNAs is further supported by the observed facts that the locations of
flagellar calmodulin mRNA and the tubulin mRNAs are
different in 70-min cells. When the cells were stained with
the anti-DIG antibody without prior hybridization with
the DIG-labeled probes or treated with RNase before the
hybridization, no staining was observed (Fig. 4 C; and data
not shown).
Involvement of Microfilaments in the Localization of DS mRNAs
Localization of mRNAs has been studied in many systems (13, 22, 26). In some of the systems, it has been suggested that translocation of mRNA requires cytoskeletal components (22). Because Naegleria amebas have an actin-based microfilament system (24) but no organized microtubule system before 50 min into the differentiation (25), we tested the possible role of the microfilament system in the localization of the DS mRNAs by using cytochalasin D, which disrupts microfilament systems in many eukaryotic cells.
Addition of cytochalasin D at the beginning of differentiation inhibited the differentiation in a dose-dependent
manner. In 20 µg/ml of cytochalasin D, 60% of the cells
formed flagella. In 50 or 100 µg/ml of cytochalasin D, the
differentiation was strongly inhibited (Fig. 5 A). In these
experiments, most of the cells changed their shape into
spheres less than 30 min after the initiation of differentiation and remained as spheres until the end of the differentiation (Fig. 6).
To better understand the nature of the inhibition of
the differentiation by cytochalasin D, we measured the
amount of the -tubulin mRNA in cytochalasin D-treated
cells. As shown in Fig. 5 B, cytochalasin D partially inhibited the transient accumulation of the mRNA. In the presence of 50 µg/ml cytochalasin D, the amounts of
-tubulin
mRNA at 20, 40, and 60 min after the initiation of differentiation were 73, 59, and 40% of that of the control cells, respectively. At 100 µg/ml of cytochalasin D, accumulation of the
-tubulin mRNA was further inhibited. Cytochalasin D also inhibited the transient accumulation of
the
-tubulin mRNA and the Class I mRNA in a similar
way (data not shown). Addition of cytochalasin D at a
later stage (80 min) had no effect on the formation of flagella (data not shown).
Because addition of cytochalasin D (50 µg/ml) at the initiation of differentiation inhibited accumulation of the tubulin mRNAs by only ~50%, we examined the effect of
cytochalasin D on the localization of the mRNA by in situ
hybridization. We added cytochalasin D at the initiation of
differentiation and examined the effect of the drug treatment on localization of the DS mRNAs. In cytochalasintreated cells (50 µg/ml), the -tubulin mRNA was found
evenly distributed or in patches in the cytoplasm of the cytochalasin-treated cells (Fig. 6). Only less than 9% of the
cells at 20 min and 5% of the cells at 70 min showed specific localization of the
-tubulin mRNA. The
-tubulin
mRNA, Class I mRNA, and flagellar calmodulin mRNA
were also found dispersed in the cytochalasin-treated cells (data not shown).
Unlike the effect of cytochalasin D, taxol (up to 50 µM) or colchicine (up to 20 mM) treatment did not cause significant effects on Naegleria differentiation when added at the beginning of differentiation.
By in situ hybridization, we have examined the distribution of four mRNAs that are accumulated transiently and
specifically during differentiation of N. gruberi; -tubulin,
-tubulin, Class I, and flagellar calmodulin mRNA. Our
findings demonstrate that these DS mRNAs are specifically localized in a limited area of the differentiating cells
at the early stages of differentiation, and then, as the differentiation proceeds, the DS mRNAs are translocated from the base of flagella to the posterior of the differentiating cells with a discrete order.
The specific localization of the four DS mRNAs became evident 20 min after the initiation of differentiation, at which time transcription of the genes was just activated (19). Until 40 min, the four DS mRNAs were colocalized, and the location was clearly distinct from that of the nucleus (see Figs. 1 and 3; Table III). However, at 70 min the location of three of the four DS mRNAs, the tubulin mRNAs and Class I mRNA, was different from that of the flagellar calmodulin mRNA (Fig. 3; Tables II and III). In >80% of flagellated cells, the tubulin mRNAs were localized at the base of the growing flagella, very close to the nucleus, where the basal bodies are located (Fig. 7) (25). On the contrary, the flagellar calmodulin mRNA was found at the base of flagella only in 16% of the flagellated cells at this stage. After this stage, the tubulin mRNAs also began to move away from the basal body area toward the posterior of the cells. By 120 min, the differentiation was completed; the DS mRNAs were still specifically localized in some cells, but the staining was very faint. In a small portion of cells, the DS mRNAs were localized at more than two areas. This result might be related to the fact that as many as 20% of cells can have more than two flagella (8).
This localization of the mRNAs could be mediated through the microfilament system. Because the amount of tubulin in the cell is very low (18) and no organized microtubular structures are observed at early stages of differentiation (20 and 40 min [25]), it is difficult to imagine that the initial transport of the DS mRNAs is mediated by microtubules. The fact that cytochalasin D treatment at the beginning of differentiation inhibited localization of the DS mRNAs is consistent with this hypothesis, but the effect of this drug on the accumulation of these DS mRNAs makes a firm conclusion impossible.
Addition of cytochalasin D (50 µg/ml) at the beginning
of differentiation caused Naegleria amebas to become
spherical more quickly than control cells. This might be a
result of the disruption of the microfilament system, the
main cytoskeletal system in Naegleria amebas. In this condition, the specific localization of the DS mRNAs, the formation of flagella and cytoskeletal microtubules, and hence
differentiation itself were almost completely inhibited. Even though cytochalasin D partially inhibited the accumulation of the DS mRNAs (~50% of the control), this
partial inhibition alone does not explain the complete prevention of the differentiation. About 50% of cells can
form flagella when actinomycin D is added at 40 min after
the initiation of differentiation, at which time the amount
of -tubulin mRNA has only reached ~50% of the maximum (9, 20). When we examined the synthesis of
-tubulin during the differentiation of cytochalasin-treated cells
(50 µg/ml, at the beginning of differentiation) and of control cells using a monoclonal antibody (25), we did not find
significant decrease (Cho, H.I., and J.H. Lee, unpublished
result).
Even though we do not understand the underlying
mechanism, this specific colocalization and presumably
translation of the DS mRNAs in one area could greatly increase the local concentration of the protein products.
This high concentration of - and
-tubulin at the area
could facilitate formation of microtubules for the basal
body. The continuous translation of the tubulin mRNAs
and flagellar calmodulin mRNA at or near the base of the
growing flagella and subsequent transportation of the proteins to the growing end (11) could also facilitate the elongation of the flagella.
The localization of the tubulin mRNAs could also be important for the formation of the CSMT. Walsh observed the CSMT radiating from the base of the flagella (25). He also showed that inhibition of protein synthesis before 52 min of differentiation inhibited formation of both flagella and CSMT. However, addition of cycloheximide between 52 and 62 min of differentiation inhibited the formation of the CSMT only. Addition of the drug after 62 min did not block formation of either organelles (25). These facts imply that further protein synthesis is required for the formation of the CSMT even after the completion of protein synthesis required for the formation of flagella and that some of the proteins are organized around the basal bodies to form a microtubule organizing center (15, 23) for the CSMT. Even though we cannot rule out the possibility that the amount of tubulin (and/or other component proteins) synthesized until 62 min of differentiation is not enough to form both organelles, these data suggest that the whole events required for the formation of flagella and CSMT are accomplished by 70 min after the initiation of differentiation (25), before the onset of translocation of the tubulin mRNAs from the base of the flagella.
The ordered translocation of the DS mRNAs, first the flagellar calmodulin mRNA then the tubulin mRNAs, to the posterior of the flagellated cells is quite intriguing. Based on the data in Table II, we may guess that the translocation of the flagellar calmodulin mRNA starts between 60 to 65 min after the initiation of differentiation. Unlike the tubulins, which are the component of the flagellar axonemes and CSMT, the flagellar calmodulin is specific for the flagellar axoneme (7), and the cellular events required for the formation of the flagella are completed before the completion of the events for the formation of the CSMT (see above). Based on these facts, we might assume that the translocation of the flagellar calmodulin mRNA begins after the completion of the cellular events for the formation of flagella, and then, after the completion of the events for the formation of the CSMT, translocation of the tubulin mRNAs ensues. These results implicate that the four DS mRNAs are transported to a specific area after being transcribed in the nucleus at early stages of differentiation and that there might be another mechanism that determines the order of translocation of different groups of mRNAs (e.g., mRNAs for microtubule proteins and mRNAs for flagellar specific proteins) toward the posterior as the differentiation proceeds. This ordered translocation of the mRNAs also suggests that this translocation is a regulated process and not a result of CSMT elongation.
The cellular function of the translocation of the mRNAs
is not clear. However, one interesting possibility is that the
translocation might be related with specific degradation of
the mRNAs in later stages of differentiation. This translocation of the DS mRNAs began just after the completion
of protein synthesis, which is required for the formation of
flagellar apparatus and CSMT (Table II and reference 25).
At the same time, the amount of the DS mRNAs began to
decrease rapidly (19, 20), and we have shown previously
that the stability of the -tubulin and Class I mRNAs is
specifically regulated in these stages of differentiation (2). It is also possible that the translocation of the localized
mRNAs at the posterior of the cells might be important
for the later stage of cell differentiation, e.g., formation of
the flagellate shape (25).
It should be noted that the Class I mRNA, which could encode a protein with a molecular mass up to 200 kD, is colocalized with the tubulin mRNAs. A few large microtubule-associated proteins have a molecular mass of 160-200 kD (1, 5, 15). We do not know the protein product of the Class I mRNA, but it is possible that the mRNA encodes a component of the basal body or of the microtubule system. If this turns out to be true, the specific localization of the DS mRNAs might give a reasonable account of a mechanism by which the formation of basal bodies and flagella, and later the formation of the CSMT, are coordinated.
Received for publication 13 January 1997 and in revised form 5 March 1997.
1. Abbreviations used in this paper: CSMT, cytoskeletal microtubules; DIC, differential interference contrast; DIG, digoxygenin; DS, differentiation-specific.We thank Drs. C. Walsh (University of Pittsburgh), D.K. Shea (Eastern Nazarene College), and B.G. Kang (Yonsei University) for constructive and critical suggestions.