Flagellar Membrane Proteins of Tetraselmis striata Butcher (Chlorophyta)
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Protist, Vol. 151, 147–159, August 2000 © Urban & Fischer Verlag
http://www.urbanfischer.de/journals/protist Protist
ORIGINAL PAPER
Flagellar Membrane Proteins of Tetraselmis striata
Butcher (Chlorophyta)
Stefan Gödel, Burkhard Becker1, and Michael Melkonian
Botanisches Institut, Universität zu Köln, Gyrhofstr. 15, 50931 Köln, Germany
Submitted December 23, 1999; Accepted May 11, 2000
Monitoring Editor: Randall S. Alberte
Highly purified flagella of the green alga Tetraselmis striata (Chlorophyta) were extracted by Triton X-
114 phase partitioning. SDS-PAGE analysis revealed that most proteins were present in the aqueous
phase, only two prominent flagellar membrane proteins (fmp) of apparent molecular weight 145 and
57 kDa (fmp145 and fmp57) were enriched in the detergent phase. Fmp145 was purified by gel perme-
ation chromatography. Glycosidase treatment in combination with lectin blot analysis showed that
fmp145 is a glycoprotein containing 3-5 N-glycans of the high mannose and/or hybrid type. A poly-
clonal antibody (anti-fmp136) was raised against the deglycosylated form of fmp145 and used to lo-
calize fmp145 by immunofluorescence and immunoelectron microscopy. Immunogold labeling
showed fmp145 to be present between the scale layers and the flagellar membrane. During flagellar
regeneration fmp145 is incorporated evenly and rapidly into the newly developing flagella. Anti-
fmp136 specifically cross-reacted with flagella of only a subgroup of Tetraselmis strains character-
ized by a specific flagellar hair type (type II according to Marin et al. 1993) and thus could be a useful
immunomarker for the identification of Tetraselmis strains by fluorescence microscopy.
Introduction
The scaly green flagellates (prasinophytes) are char- scales”, double scales) that cover the pentagonal
acterized by the presence of the cell-surface associ- scales, and flagellar hairs (hair-scales) that lie in two
ated structures of distinct size and shape termed rows on opposite sides of the flagellum. A fourth
scales (Becker et al. 1994; Sym and Pienaar 1993). type of flagellar scale, the “knotted scales”, has
In most prasinophytes scales can be found on the been found in several strains of Tetraselmis (Becker
cell body as well as on the flagella. In the Chloroden- et al. 1990), but their precise arrangement on the
drales (the genera Tetraselmis and Scherffelia) the flagella and their distribution within the Chloroden-
cell body is covered by a cell wall, which represents drales are not known.
a periplast of fused scales, commonly known as the Prasinophyte scales consist mainly of acidic
theca. The four flagella of a cell are covered by dif- polysaccharides and, as minor components, several
ferent types of scales (Becker et al. 1990): Pentago- glycoproteins that vary in number and quantity be-
nal scales (underlayer scales) are attached to the tween different scale types (reviewed in Becker et al.
flagellar membrane, rod-shaped scales (“man 1994). Some scale types (e.g. the rod-shaped
scales) seem to contain no proteins (Becker et al.
1
Corresponding author;
1990). It is thought, therefore, that the carbohy-
fax 49-221-4705181 drates determine the structure of the different scale
e-mail b.becker@uni-koeln.de types, whereas the glycoproteins provide linkages
1434-4610/00/151/02-147 $ 12.00/0148 S. Gödel et al.
between subunits of scales (Becker and Melkonian
1992) and between underlayer scales and the flagel-
lar membrane (Becker et al. 1996).
Scales show a remarkable diversity in ultrastruc-
ture among prasinophytes, and for this reason they
are being widely used for taxonomic purposes (sum-
marized in Sym and Pienaar 1993). In particular, the
hair-scales have been shown to exhibit a great intra-
and intergeneric variation (Marin and Melkonian
1994; Marin et al. 1993). Based on the structure of
the flagellar hairs, Tetraselmis strains were grouped
into 4 clusters (Marin et al. 1993). An investigation of
the glycosylation pattern of scale-associated pro-
teins in Tetraselmis using lectins showed group spe-
cific differences in the glycosylation pattern (Becker
et al. 1995). Binding of the lectin PNA (peanut agglu-
tinin) was restricted to a group of Tetraselmis
species, characterized by the presence of type I flag-
ellar hairs (according to Marin et al. 1993).
In this contribution we describe the isolation and
biochemical characterization of a major flagellar Figure 1. Light microscopy of Tetraselmis striata (A)
membrane glycoprotein of Tetraselmis striata. Using and the isolated flagella fraction (B). Note the absence of
a polyclonal antibody directed against the deglyco- thecae, cells or cell fragments in (B). The proximal end of
sylated form of this protein, we show that the epi- flagella is characterized by a swelling (arrows). Nomarski
topes recognized by the antibody are found only in interference contrast images. Scale bar: 10 µm.
Tetraselmis species carrying type II flagellar hairs.
Results
Isolation of Flagella
Purified flagella are a prerequisite for the isolation of
flagellar membrane proteins. Flagella (Fig. 1B) were
isolated from Tetraselmis striata (Fig. 1A) as de-
scribed in Methods. The preparation was essentially
free of contamination by thecae, cell bodies or cell
debris (Fig. 1B). As shown previously, flagella iso-
lated in this way retain their typical ultrastructure,
the flagellar membrane completely encircling the ax-
oneme (Becker et al. 1990). The proximal end of iso- Figure 2. SDS-PAGE analysis (4–12%) of the purifica-
lated flagella is characterized by a swelling (Fig. 1B, tion of the major flagellar membrane protein fmp145
arrowheads), presumably a result of partial depoly- using Triton X-114 phase partitioning and gel perme-
merisation of the axoneme due to calcium influx into ation chromatography. Lane 1 total flagellar proteins.
the flagella during pH-shock. Lane 2 Triton X-114-insoluble fraction of flagellar pro-
teins. Lane 3 Triton X-114-soluble fraction. Lane 4
aqueous phase of the Triton X-114 phase partitioning.
Isolation of the Major Flagellar Proteins Lane 5 detergent phase of the Triton X-114 phase parti-
Isolation of flagellar membrane proteins was moni- tioning. Lane 6 purified fmp145 fraction after gel perme-
ation chromatography. The positions of the major flag-
tored by protein determination and SDS-PAGE anal-
ellar membrane proteins [145 kDa (fmp145) and 57 kDa
ysis (4–12% gradient gel) followed by densitometric (fmp57)] are marked by arrowheads. Molecular weight
analysis of the fractions obtained (see Methods for markers are indicated on the left from top to bottom:
details). Flagella were solubilized in 1% (w/v) Triton 205, 116, 97, 66, 45 and 29 kDa, respectively. All lanes
X-114 (Fig. 2, lane 1) and the detergent-insoluble were loaded with 10-12 µg protein except lane 5 and 6
fraction (Fig. 2, lane 2) was separated from the de- which were loaded with 2 µg.Flagellar Membrane Proteins of Tetraselmis 149
tergent-soluble fraction (Fig. 2, lane 3) by centrifuga- to determine the relative amount of fmp145 within
tion. The supernatant (Fig. 2, lane 3) contained 85% this fraction. The number of fmp145 molecules was
of the total flagellar proteins, including water soluble found to be about 3000 per flagellum.
proteins of the extra- and intraflagellar matrix, mem-
brane proteins and axonemal proteins (e.g. tubulin
50 kDa, Fig. 2, lane 3). At least 40 Coomassie Blue-
Fmp145 is a Glycoprotein Containing N-gly-
stained polypeptide bands were detected in 1D
cans of High Mannose and/or Hybrid Type
SDS-PAGE gels of the detergent-soluble fraction Because staining of fmp145 after SDS-PAGE with
(Fig. 2, lane 3). Periodic Acid Schiff’s reagent (PAS-stain) indicated
Further fractionation of the detergent-soluble pro- that fmp145 is a glycoprotein (data not shown), we
teins into a detergent (Triton X-114) and aqueous investigated its glycosylation pattern. Treatment of
phase by phase partitioning was performed to iden- fmp145 with glycosidases (Fig. 3A) and lectins (Fig.
tify those proteins likely to be tightly associated with 3B) confirmed that fmp145 is a glycoprotein. To
the flagellar membrane. Analysis of the fractions by evaluate the nature of the glycans present on
SDS-PAGE analysis revealed that most polypep- fmp145, N-glycans were enzymatically cleaved
tides (81% of the detergent-soluble fraction) re- using N-Glycosidase F or Endoglycosidase H prior
mained in the aqueous phase after two extractions to SDS-PAGE analysis. Both glycosidases caused a
with Triton X-114 (Fig. 2, lane 4), whereas only two molecular weight shift of 9 kDa (Fig. 3A, lanes 1–4)
major proteins with apparent molecular weights of corresponding to a carbohydrate content of 6%.
145 and 57 kDa plus several minor protein bands Whereas N-Glycosidase F cleaves most known N-
were recovered in the detergent phase (16% of the glycosidic glycan types (with the exception of com-
detergent-soluble fraction; Fig. 2, lane 5). Based on plex glycans containing fucose linked α-1→3 to the
densitometric analysis, the two major flagellar mem- core chitobiose unit; Tarentino and Plummer 1994),
brane proteins account for at least 75% (145 kDa Endoglycosidase H treatment results in cleavage of
35% and 57 kDa 40%) of the proteins in the deter- the core chitobiose unit and occurs only in high-
gent phase. Taking into account the apparent mannose and hybrid type glycans (Tarentino and
molecular weights of these two proteins, a molecu- Plummer 1994). Since the same molecular weight
lar ratio of 1:3 was calculated. shift was observed for N-Glycosidase F or Endogly-
cosidase H -treated fmp145 (Fig. 3A, lane 2 and 4),
high mannose and/or hybrid type, but not complex
Purification of the 145 kDa Flagellar type glycans, are present on fmp145.
Membrane Protein (fmp145) Further investigation of the glycan structures of
For further purification of fmp145, the Triton X-114 fmp145 was performed using lectin blot analysis in
concentration in the detergent phase was reduced combination with N-Glycosidase F treatment (Fig.
from 4% to 0.07% (w/v) using Bio-beads SM (see 3B). Of eight different lectins tested, only GNA, WGA
Methods). To improve solubility, SDS was added and DSA (Fig. 3B, GNA, WGA, DSA, lane 1) bound to
(final concentration 0.05% [w/v] SDS) and the pro- fmp145. After enzymatic cleavage of N-glycans with
teins separated by gel permeation chromatography N-Glycosidase F, lectin labeling was abolished (Fig.
under non-reducing conditions. Fmp145 elutes with 3B, GNA, WGA, DSA, lane 2). The N-Glycosidase F-
an apparent molecular mass of 145 kDa. The native treated form of fmp145 also shows no PAS-staining
flagellar membrane protein (fmp145) is, therefore, (data not shown). Based on these results, fmp145
present as a monomer under the conditions tested. presumably contains only N-glycosidic but no O-gly-
SDS-PAGE analysis of the fmp145 fraction (Fig. 2, cosidic glycans. Therefore, N-Glycosidase F-treated
lane 6) revealed that most Triton X-114 extractable fmp145 (fmp136) represents the completely degly-
proteins were removed, especially the second major cosylated polypeptide part of the glycoprotein. The
protein component (fmp57). Densitometric analysis lectin DSA is specific for galactose linked β-1→4 to
of the Coomassie Blue-stained gel showed that N-acetylglucosamine or for terminal N-acetylglu-
fmp145 accounts for about 95% of the polypeptide cosamine (Wu and Sugii 1991). Since no RCA signal
present in the final fraction and is at least 200-fold (specific for terminal galactose; Wu and Sugii 1991)
enriched compared to the detergent-solubilized was detected, DSA probably recognized N-glycans
flagellar proteins (Fig. 2, lane 1). of the hybrid type containing terminal N-acetylglu-
The number of fmp145 molecules per flagellum cosamine. These data are supported by the positive
was estimated using the number of harvested flag- WGA labeling indicating terminal chitobiose units
ella, the protein content of the isolated flagella and (Wu and Sugii 1991). Given these results and the
desitometric analysis of the isolated flagella fraction GNA binding which is specific for terminal manno-150 S. Gödel et al. Figure 3. Analysis of the glycoprotein nature of fmp145. (A) Purified flagellar membrane protein fmp145 (lane 1 and 3) was treated with N-Glycosidase F (lane 2) or Endoglycosidase H (lane 4), separated by 5% SDS-PAGE and stained with Coomassie Brilliant Blue. Protein load was 2 µg in each lane. Molecular weight markers are indicated on the left, from top to bottom: 205, 116 and 97 kDa, respectively. (B) Purified fmp145 (lane 1) or N-Glycosidase- treated fmp145 (fmp136, lane 2) were separated by SDS-PAGE (4–12% gel), and transferred to a PVDF membrane. Different portions of the blot were stained with Amido-black (AB) or probed with wheat germ agglutinin (WGA), Datura stramonium agglutinin (DSA) and Galanthus nivalis agglutinin (GNA). Protein load was 1 µg protein per lane. The position of the glycosylated (fmp145) and deglycosylated (fmp136) form of the flagellar membrane protein is marked by arrowheads. Molecular weight markers are indicated on the right, from top to bottom: 205, 116, 97 and 66 kDa, respectively. Figure 4. Immunoblot analysis using anti-fmp136. Purified fmp145 (A) and N-Glycosidase F-treated fmp145 (fmp136, B), isolated flagella (C) and a crude cell fraction of Tetraselmis striata (D) were separated on a 4–12% gra- dient gel and transferred to a PVDF membrane. Lane 1 Amido-black-stained portion of the blots. Separated pro- teins were probed with the anti-fmp136 IgG (10 µg/mL, lane 2) or preimmune IgGs (lane 3) and visualized using an anti-rabbit IgG alkaline phosphatase conjugate using the NBT/X phosphate system. Protein load was 1 µg in A and B and 10 µg in C and D. The position of the marker proteins is indicated, from top to bottom: 205, 116, 97, 66 and 45 kDa, respectively. The single antigenic band of the glycosylated fmp145 or deglycosylated fmp136 form of the flagellar membrane protein detected by anti-fmp136 is marked by arrowheads.
Flagellar Membrane Proteins of Tetraselmis 151
biose (Shibuya et al. 1989), fmp145 presumably con- IgGs or preimmune IgGs as control and visualized
tains 3-5 N-glycans (estimated by the molecular shift using a secondary anti-rabbit IgG-FITC conjugate. A
of 9 kDa) of the high mannose type and/or the hybrid strong specific fluorescence signal of only the four
type containing terminal chitobiose. flagella (Fig. 5A–D) was observed. The fluorescence
of the flagella was uniform (see also Fig. 6), but
sometimes a punctate pattern was observed. Fluo-
A Polyclonal Antibody Against fmp145 rescence of the cell body was never seen.
Plant carbohydrates are often highly antigenic (Faye These data were corroborated by immunogold
and Chrispeels 1988) and interfere with the produc- electron microscopy of cells of Tetraselmis striata
tion of specific polypeptide antibodies. Therefore, (postembedding labeling). A strong labeling of the
the flagellar membrane protein fmp145 was degly- area between flagellar membrane and the scales
cosylated using N-Glycosidase F yielding fmp136. (Fig. 5E and F) with gold particles was observed.
The anti-fmp136 antibody recognized on Western Few gold particles labeled the flagellar membrane
blots, the glycosylated as well as the deglycosylated region directly. Controls using preimmune IgGs
form of fmp145 (Fig. 4A and B). The antibody also showed no labeling at all (Fig. 5F). No labeling of the
detected fmp145 in an isolated flagella fraction (Fig. cell body was found (data not shown), indicating
4C) and a crude cell extract (Fig. 4D). Binding of the that fmp145 is restricted to the flagella in interphase
antibody to fmp145 by anti-fmp136 is not abolished cells.
by periodate treatment of blots (data not shown).
These results indicate that anti-fmp136 is specific
for peptide epitopes.
Fmp145 is Synthesized During Flagellar
Regeneration
Cells of Tetraselmis striata were deflagellated, fixed
Immunolocalization of fmp145 at different times during flagellar regeneration and
For immunolocalization of fmp145, cells of Tetrasel- analyzed by immunofluorescence microscopy using
mis striata were fixed, incubated with anti-fmp136 anti-fmp136 (Fig. 6). Approximately 80–90% of the
Figure 5. Immunolocalization of fmp145 using immunofluorescence and immunogold electron microscopy. (A and
C) Nomarski interference contrast image corresponding to the immunofluorescence images (B and D). (A und B)
anti-fmp136 IgG, (C and D) preimmune IgG. Scale bar: 10 µm. (E and F) Immunogold electron microscopy using
anti-fmp136 (E) or preimmune IgG (F). Cells were fixed in 0.25% (w/v) glutaraldehyde/ 3% (w/v) paraformaldehyde
and embedded in Lowicryl K4M as described in Methods. Scale bar: 0.5 µm.152 S. Gödel et al.
cells regenerated their flagella. In cells fixed immedi- reached about 8 µm (Fig. 6F) . The fluorescence sig-
ately after deflagellation, a fluorescence signal con- nal is distributed uniformly on the flagella with the
sisting of two spots is visible at the basis of the flag- exception of a less immunoreactive space of
ellar pit (Fig. 6B). These two spots remain visible 0.5–1.5 µm between the flagella and the two im-
during the whole flagellar regeneration. Thirty min- munoreactive spots (Fig. 6).
utes after deflagellation small fluorescent flagella
were observed within the flagellar groove (Fig. 6C).
Over the next few hours the cells regenerate new
Anti-fmp136 Cross Reacts With
flagella (Fig. 6D–F). Flagellar growth was followed up
Homologous Proteins Only in Closely
to two hours after deflagellation, when the flagella
Related Tetraselmis Strains
The genus Tetraselmis has previously been grouped
into four major clusters ( I–IV) on the basis of flagellar
hair ultrastructure (Marin et al. 1993). To test
whether anti-fmp136 raised against the deglycosy-
lated form of fmp145 of Tetraselmis striata (type II
flagellar hairs), cross reacts with flagellar membrane
proteins in other taxa, 13 additional Tetraselmis
strains displaying flagellar hair types I-IV were inves-
tigated by immunofluorescence and western blot-
ting (Table 1 and Fig. 7). As an example, the im-
munofluorescence pattern of one member of each
group is shown in Fig. 7. In the Tetraselmis species
displaying type II flagellar hairs the epitope recog-
nized by anti-fmp136 was detected by immunofluo-
rescence and western blots (Fig. 8). In contrast, we
were not able to detect any signal in all other tested
strains (Table 1, Fig. 8). Using immunofluorescence
some strains showed a weak fluorescence of the
flagellar pit (two Tetraselmis strains) or the whole
flagella (Scherffelia), but since the preimmune serum
yielded a similar fluorescence and no cross-reactiv-
ity on western blots was found (Fig. 8), the fluores-
cence is most likely nonspecific. No specific cross-
reactivity with anti-fmp136 was observed in Scherf-
felia dubia, the other genus in the Chlorodendrales
(thecate prasinophytes), in Nephroselmis pyriformis,
which displays similar flagellar scales as Tetra-
selmis, and in the naked chlamydomonad Dunaliella
bioculata. In conclusion, specific cross-reactivity
using anti-fmp136 was only observed in Tetraselmis
strains characterized by the presence of flagellar
hairs of type II (4 strains analyzed). These strains ex-
hibited a uniform fluorescence signal on their four
flagella and showed a single antigenic 145 kDa
polypeptide band in western blots.
Discussion
We have isolated a major flagellar membrane protein
Figure 6. Indirect immunofluorescence of cells of
Tetraselmis striata during flagellar regeneration using (termed fmp145) from a purified flagella preparation,
anti-fmp136. Cells were fixed before (A), immediately using Triton X-114 phase partitioning and gel per-
after deflagellation (B), 30 min (C), 60 min (D), 90 min (E) meation chromatography. Fmp145 is a glycoprotein
and 120 min (F) after deflagellation and processed for carrying high-mannose/hybrid N-glycans. An anti-
immunofluorescence microscopy. Scale bar: 10 µm. body raised against the deglycosylated form ofFigure 7. Indirect immunofluorescence using anti-fmp136 of Tetraselmis strains displaying flagellar hair types I–IV. Nomarski interference contrast and corresponding immunofluorescence probed with anti-fmp136 or preimmune IgG are shown. (A–D) Tetraselmis hazenii (type I flagellar hairs), (E–H) Tetraselmis suecica (type II), (I–L) Tetraselmis verrucosa (type III) and (M–P) Tetraselmis astigmatica (type IV). On the left the anti-fmp 136 IgG are shown, on the right the corresponding preimmune control. Scale bar: 10 µm.
Figure 8. Immunoblot analysis using anti-fmp 136. Isolated flagella from different strains of Tetraselmis and Scherffelia were separated on a 4-12% gradient gel and transferred to PVDF membrane. First Row: Strains display- ing type I flagellar hairs. Second Row: Strains displaying type II flagellar hairs. Third Row: Strains displaying type III flagellar hairs. Lane 1, amido black stain of a portion of the blot. Separated proteins were probed with the anti- fmp136 IgG (10 µg/mL, lane 2) or preimmune IgGs (lane 3) and visualized using an anti-rabbit IgG alkaline phos- phatase conjugate using the NBT/X phosphate system. The position of the marker proteins is indicated on the left, from top to bottom: 205, 116, 97, 66 and 45 kDa, respectively.
Flagellar Membrane Proteins of Tetraselmis 155
Table 1. Presence of epitopes recognized by anti-fmp145 in various green algae.
Flagellar Strain2 Species Source Immunofluorescence Western blot
hair type1 ––––––––––––––––––––– –––––––––––––––––––––––
Immune preimmune immune preimmune
I M0568 T. chui SAG 8–6 – – – –
M0607 T. hazenii CCMP 891 – – – –
M0583 T. tetrathele Ply 272 – – n.d. n.d.
M0795 S. dubia SAG 40.89 + + – –
II M0590 T. apiculata CCMP 878 ++ – + –
M0610 T. levis CCMP 895 ++ – + –
M0593 T. suecica CCMP 907 ++ – + –
M0580 T. striata Ply 443 ++ – + –
III M0806 T. marina CCMP 898 – – n.d. n.d.
M0836 T. spec CCMP 973 + + – –
M0629 T. verrucosa CCMP 918 + + – –
M0612 T. verrucosa/rubens CCMP 919 – – n.d. n.d.
IV M0833 T. astigmata CCMP 880 – – n.d. n.d.
M0579 T. cordiformis SAG B 26.8 – – n.d. n.d.
M0832 T. convoluta – – n.d. n.d.
M0773 N. pyriformis CCAP 1960/3 – – n.d. n.d.
M0718 D. bioculata SAG 19–4 – – n.d. n.d.
++ strong fluorescence of whole flagella, + a week fluorescence or positive signal in western blots using anti-
fmp136, – no signal, n.d. not determined. Sources of strains: SAG Sammlung von Algenkulturen, Albrecht-Haller In-
stitut für Pflanzenwissenschaften, Universität Göttingen, Göttingen, Germany; CCAP Culture Collection of Algae
and Protozoa, Dunstaffage, Scotland; CCMP Provasoli-Guillard Center for Culture of Marine Phytoplankton, West
Boothbay Harbor, Maine, USA; Ply Plymouth Culture Collection, Mar. Biol. Assoc. U. K., The Laboratory, Citadell
Hill, Plymouth, U.K. 1 according to Marin et al. (1993), 2 strain numbers in the culture collection of algae at the Uni-
versity of Cologne (CCAC), Germany.
fmp145 (anti-fmp136) was used to localize fmp145 the situation in other algal systems (e.g. Bloodgood
to the flagellar membrane/scale layer by immuno- 1990; Bouck et al. 1990). The presence of fmp145 in
gold labeling. Fmp145 appears to be synthesized the detergent phase after Triton X-114 phase parti-
during flagellar regeneration, since immunofluores- tioning indicates that it strongly bound to the flagel-
cence microscopy demonstrated the incorporation lar membrane, but it does not rule out the possibility
of fmp145 into the growing flagella, and no fmp145 that it is either a peripheral protein or is directed into
could be detected intracellularly before deflagella- the detergent phase by interaction with another
tion using immunogold electron microscopy. In this membrane protein (e.g. fmp57). The immunogold lo-
respect, fmp145 differs from the scale-associated calization may support the latter possibilities, since
proteins (SAPs) of Scherffelia dubia which have the epitopes recognized by anti-fmp136 seem to be
been shown not to be synthesized during flagellar mostly confined to the area between the flagellar
regeneration, but instead they are recruited from a membrane and the scale layers (see Results). Inter-
pool mainly localized at the plasma membrane estingly, we could not detect fmp145 labeling of the
(Perasso et al. 2000). cell body plasma membrane, indicating that there is
We calculated the number of fmp145 molecules either a barrier between the two membrane domains
to be about 3000 per flagellum. Interestingly, the (as in Chlamydomonas reinhardtii; Bloodgood
number of pentagonal scales per flagellum is about 1990), the diffusion of the protein is inhibited by
the same (McFadden and Melkonian 1986a; Melko- other constraints (e.g. interaction with the ax-
nian 1982). Whether this is just a coincidence or re- oneme), or the epitopes are hidden or blocked in the
flects some specific relationship (e.g. fmp145 could intact cell.
be the flagellar membrane receptor for the pentago- Using immunofluorescence we observed a label-
nal scales) remains to be determined. ing of the flagella only when fixed cells were used.
The number of major flagellar membrane proteins When live cells were incubated with the antibody no
in Tetraselmis seems to be low. This is very similar to fluorescence of the flagella was observed (unpub-156 S. Gödel et al.
lished observations). Most likely the flagellar scales bodies. For light microscopical documentation, cells
present an impenetrable barrier to the antibody, and and flagella were fixed for 20 min in 3% (w/v)
it is only upon fixation that the epitopes become ac- formaldehyde and 0.25% (v/v) glutaraldehyde final
cessible. Obviously fixation alters the arrangement concentration in culture medium at room tempera-
of the flagellar scales (Melkonian 1982). ture.
The most surprising finding using anti-fmp136 Isolation of flagellar membrane proteins using
was that the recognized epitopes seem to be re- Triton X-114 phase partitioning: The isolated flag-
stricted to a certain subgroup of Tetraselmis strains. ella suspension (10 mL) was mixed with 10 mL ASP-
Although all strains of Tetraselmis investigated and 2 [containing: 2% (v/v) Triton X-114, 0.06 % (w/v)
Scherffelia dubia reveal, upon SDS-PAGE, a protein NaN3, Leupeptin 1µg/mL] and incubated for 60 min
of similar molecular weight in flagellar membrane or with continuous stirring on ice. Subsequently, the
flagella fractions, only proteins of strains of Tetra- solution was centrifuged (47,800 g, 30 min, 4 °C;
selmis with type II flagellar hairs were specifically Sorvall RC 28S; SS34 rotor) to remove detergent-in-
recognized. Strains of Tetraselmis have been soluble material. Phase partitioning was performed
grouped into 4 major clusters based on the ultra- with the detergent-soluble fraction modified accord-
structure of flagellar hairs (Marin et al. 1993). These ing to the method of Bordier (1981) and Pryde
clusters are supported by other ultrastructural char- (1986). Aggregation of micelles was induced by in-
acters and by molecular phylogenetic analysis cubation of the solution for 5 min at 30 °C. Centrifu-
based on sequence comparisons of the rRNA gation (5800 g, 10 min, 30 °C; Sorvall RC 28S, SS34
operon and represent monophyletic lineages within rotor) yielded an upper aqueous and a lower deter-
the genus (Marin and Melkonian unpublished re- gent phase. The aqueous phase was carefully re-
sults). Anti-fmp145 thus represents a novel immuno- moved and again incubated for 20 min on ice with
marker for the identification of a subgroup of Triton X-114 [final concentration 0.5% (v/v)] followed
Tetraselmis strains by immunofluorescence mi- by phase partitioning as described above. The two
croscopy. detergent phases were collected and washed with
ASP-2 medium saturated with Triton X-114. For re-
moval of the detergent, the detergent phase was di-
Methods luted with 10 mL 20 mM sodium phosphate buffer
(150 mM NaCl, pH 7.2) and placed on ice until deter-
Strains and culture conditions: The origin of the gent micelles disappeared. Removal of the Triton X-
Tetraselmis strains used in this study is listed in 114 detergent was achieved by incubation of the so-
Table 1. Marine strains were cultured in artificial sea lution with 5 g Bio Beads SM-2 (BioRad, Munich,
water (ASP-2) as described by Becker et al. (1990). Germany) for 5 h on ice with continuous stirring. Re-
Freshwater strains were cultured in modified WARIS placement of Triton X-114 from the solution was
solution (WEES-H, McFadden and Melkonian monitored with a Shimadzu UV-260 spectropho-
1986b). Cells were grown at 15 °C in 1 L Erlenmeyer tometer (Holloway 1973). Bio Beads were finally
flasks at 70 µE/m2s (Osram 36 W/25 and 36 W/30) in separated from the solution by filtration. To keep
a 14/10 h, light/dark cycle, continuously stirred and flagellar membrane proteins in solution, SDS was
bubbled with 1 L air/min. Mass cultures of Tetrasel- added to a final concentration of 0.05% (w/v).
mis striata were grown according to Becker et al. Gel-permeation chromatography (FPLC): Solu-
(1990). bilized flagellar membrane proteins were separated
Isolation of flagella: Cell culture (54 L; cell den- on a Hiload Superdex G-200pg 16/60 column using
sity: 1 × 106) were concentrated to 1.2 L using a tan- a Pharmacia FPLC system. Samples were filtered
gential flow filtration system (Pellicon, Millipore, through a Millipore membrane filter (Type HA, 0.45
Eschborn/ Germany; HVLP filter, 0.45 µm pore size). µm) prior to injection. Membrane proteins were
All further isolation steps were performed at 4 °C. eluted with 20 mM sodium phosphate buffer (150
Cells were deflagellated using pH-shock (Witman et mM NaCl, 0.05% SDS (w/v), pH 7.2) at a flow rate of
al. 1972). Cell bodies were sedimented by low- 1.5 mL/min, and monitored at 276 nm with a Uvicord
speed centrifugation (250 g, 10 min; Sorvall RC 28S; SII (Pharmacia). Fractions of the flagellar membrane
GSA rotor). The supernatant containing the flagella protein fmp145 were collected, exhaustively dia-
was centrifuged at 5000 g, 60 min (GSA rotor). The lyzed, freeze dried and stored at –20 °C.
pellet consisting of flagella, thecae and a few cell Glycosidase treatment: Prior to deglycosylation
bodies was resuspended in 10 mL culture medium with N-Glycosidase F or Endoglycosidase H (both
and subjected to low speed centrifugation (200 g, 5 from Boehringer Mannheim, Germany), lyophilized
min; Heraeus Labofuge I) to pellet remaining cell flagellar membrane protein (fmp145) was extractedFlagellar Membrane Proteins of Tetraselmis 157 with chloroform/methanol according to the method water and the nitrocellulose strips solubilized in of Wessel and Flügge (1984). The protein pellet was 300 µL dimethyl sulfoxide. For the first injection, the resuspended in 20 mM sodium phosphate buffer solution containing 4–6 µg antigen was mixed with containing 0.5% (w/v) SDS, pH 7.2 to a final con- 300 µL complete Freund’s adjuvant. Booster injec- centration of 1–1.5 mg protein/mL and the sample tions were given 19 and 43 days later with incom- boiled for 2 min. For N-Glycosidase F treatment, a plete Freund’s adjuvant and 8–15 µg antigen. Preim- threefold volume of 20 mM sodium phosphate mune serum was taken from the rabbit immediately buffer pH 7.2 [containing 50 mM EDTA, 0.05% (w/v) before the first injection was given. IgG fractions NaN3 and 0.5% (w/v) octylglucoside] was added were purified from preimmune and immune serum and the sample boiled again for 2 min. After cooling, (anti-fmp136, after the second boost) by two ammo- the sample was incubated with a final concentration nium sulfate precipitations (1.75 M) and affinity chro- of 0.4 U N-Glycosidase F and 1µM Leupeptin at 37 matography using a Protein A-Superose column HR °C overnight. For Endoglycosidase H treatment, the 10/2 (Pharmacia) and standard protocols. chloroform/ methanol-extracted protein pellet was Immunoblotting: Whole cell extract, purified resuspended in 100 mM sodium acetate buffer (pH flagella and glycosylated or deglycosylated fmp145 = 5.5) containing 0.02% (w/v) SDS and after boiling were separated by SDS-PAGE and transferred to a diluted with an equal volume of the buffer without PVDF membrane. The membrane strips were SDS. The sample was boiled and finally incubated blocked by incubation with phosphate buffered with 50 mU Endoglycosidase H, 1µM Leupeptin at saline (150 mM NaCl, 10 mM, sodium phosphate 37 °C overnight. buffer pH 7.4; PBS)/3% (w/v) bovine serum albumin SDS-PAGE and protein blotting: SDS-PAGE (BSA; RIA-grade, Sigma) overnight. The blot was was carried out following the procedure of Laemmli then incubated with the primary antibody anti- (1970) in a Mini-Gel-apparatus using gradient fmp136 or preimmune IgGs [10 µg IgG/mL in (4–12%) slab gels. Samples were heated (100 °C, 2 PBS/3% (w/v) BSA] for 90 min, washed five times min) in SDS-sample buffer, containing 0.5 % (v/v) for 15 min in PBS/0.05% (v/v) Tween 20 and blocked mercaptoethanol. Gels were stained with Coo- again for 2 h before subsequent incubation with the massie Brilliant Blue or transferred for protein blot- secondary antibody [anti-rabbit IgG alkaline phos- ting as described previously (Grunow et al. 1993). phatase conjugate, dilution 1:1000 in PBS/3% (w/v) Molecular weight standards (MW-SDS-200) were BSA; Boehringer, Mannheim]. After washing the obtained from Sigma (Deisenhofen, Germany). membrane strips five times (see above) the mem- Lectin blots: Electrophoretically transferred pro- brane was stained using the NBT/X-phosphate sys- teins were analyzed on poly-vinylidene-difluoride tem (Boehringer, Mannheim). (PVDF) membranes for lectin binding with the glycan Protein determination: Protein concentrations differentiation kit of Boehringer (Mannheim, Ger- were determined using the procedure of Neuhoff et many) according to the procedure of Becker et al. al. (1979) and BSA (fraction V; RIA grade, Sigma) as (1993). The following digoxigenin-labeled lectins standard protein. were used (for carbohydrate specificity of the lectins Time course of flagellar regeneration: For flag- see respective references): AAA (Aleuria aurantia ag- ellar regeneration, algae were harvested 3 h after the glutinin; Debray and Montreuil 1989), GNA (Galan- onset of the light regime. Flagella were amputated thus nivalis agglutinin; Shibuya et al. 1989), MAA by pH-shock and flagellar regeneration was moni- (Maackia amurensis agglutinin; Wang and Cum- tored according to Reize and Melkonian (1987) in mings 1988), RCA (Ricinus communis agglutinin; the darkness with aeration at 25 °C. Samples (1 mL) Wu and Sugii 1991), DSA (Datura stramonium agglu- of cells regenerating flagella were taken at different tinin; Wu and Sugii 1991), PNA (peanut agglutinin; time points (t before, t0, t30, t60, t90, and t120), and biosyn- Wu and Sugii 1991) and WGA (wheat germ agglu- thetic activity was immediately stopped by adding tinin; Wu and Sugii 1991). Control glycoproteins for 10 µL NaN3 (10%, w/v) and 1 µL cycloheximide the different lectins were obtained from the kit. (2 mg/mL). Algae were sedimented (Biofuge B, Production of a polyclonal antibody against Hereaus, step 1, 5 min) and fixed immediately as de- the deglycosylated fmp145: The flagellar mem- scribed below. brane protein fmp145 was deglycosylated using N- Indirect immunofluorescence microscopy: Glycosidase F, separated on preparative 4–12% Marine algae were sedimented (Labofuge 1, 80 g, gradient gels, blotted onto nitrocellulose and visual- 5 min) and carefully resuspended in 0.5 mL ASP-H ized by staining with 0.2% (w/v) Ponceau S in 3% medium McFadden and Melkonian (1986b) contain- (v/v) acetic acid. The 136 kDa band (deglycosylated ing 5 mM EDTA but no CaCl2. Immediately there- form of fmp145) was excised, destained in distilled after, cells were fixed for 20 min by adding an equal
158 S. Gödel et al.
volume of modified ASP-H containing 6% (w/v) Acknowledgements
paraformaldehyde and 0.5% (v/v) glutaraldehyde.
Freshwater algae were prepared in the same man- This study was supported by the Deutsche For-
ner with the exception that WEES-H medium Mc- schungsgemeinschaft.
Fadden and Melkonian (1986b) instead of modified
ASP-H medium was used. After complete removal
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