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The fine structure of the flagellar apparatus of Chlamydomonas moewusii
619 THE
FINE
STRUCTURE
OF THE
FLAGELLAR
CHLAMYDOMONAS SARAH
P. GIBBS,l
Marine
Laboratory,
OF
MOEWUSII
R. A. LEWIN
Biological
APPARATUS
and D. E. PHILPOTT
Woods Hole, Mass., U.S.A.
Received September 10, 1958
AN electron-microscope
examination of the fine structure of the flagellar apparatus of Chlamydomonas moewusii was carried out to support studies on the genetic control of motility in this flagellate [2]. These observations of sectioned material extend previous studies with shadowed whole mounts [3]. Methods.-Cells grown in bacteria-free culture [2] were fixed for 30 minutes in 1 per cent osmium tetroxide buffered with acetate-Verona1 to pH 7.6, washed, dehydrated quickly in an ethanol series, and imbedded in a mixture OF4 parts n-butyl and I part methyl methacrylate. Sections were cut on an experimental microtome [4] and observed with an RCA electron microscope, model EMU-2B. Results.-Each flagellum consists of a cylinder of 9 double peripheral fibrils and a central pair, all imbedded in a structureless matrix and bounded by a membrane, about 100 w thick, continuous with the plasma membrane of the cell (Fig. 1). Each individual fibril appears tubular and is loo-150 A in diameter. The 2 central fibrils project distally about 200 rnp beyond the ends of the peripheral fibrils to form a mucronate tip to the flagellum. Within the cell, the 9 double peripheral fibrils continue to form the basal body of the flagellum. As in most Protista, a cross section through the basal body appears as an electron-dense ring comprising 9 denser areas (Fig. 6). A median longitudinal section of the basal region of the flagellum reveals an H-shaped structure not previously reported in other flagella or cilia (Fig. 4). This consists of an electron-dense cylinder, about 70 rnp in diameter and 120 rnp long, lying within the peripheral fibrils and traversed slightly below its middle by a diaphragm which extends across the flagellum. Fig. 3 shows a cross section through this central cylinder, presumably cut above the transverse diaphragm since the peripheral fibrils are still fairly distinct. The central fibrils appear to end unattached just above the distal end of the cylinder and do not continue into its lumen. The paired flagella arise from opposite sides of an anterior papilla and pass to the anterior through a tunnel in the thickened cell wall. Along the median axis the two blepharoplasts join to share a common lumen (Fig. 5). Extending anteriorly into the papilla from the fused basal bodies is a thick-walled, bluntly-conical projection, whose lumen is at least partly separated from that of the blepharoplasts by one or two transverse partitions (Fig. 5). In some median sections a pair of double membranes, possibly indicating tubules or cisternae, are seen to arise together at the midpoint of the fused blepharoplasts and extend backward, one on each side of the contractile vacuole. No connection with the nucleus could be established. 1 Present address: Biological
Laboratories,
Harvard
University,
Cambridge, Experimental
Mass., U.S.A. Cell Research 15
Sarah P. Gibbs, R. A. Lewin and D. E. Philpoff
Fig. l.-Cross section through a flagellum of a normal cell. x 106,000. Fig. 2.-Cross section through a flagellum of paralysed mutant #1043. x 97,000. Fig. 3.-Cross section through the central cylinder. x 72,000. Fig. 4.-Median section through the central cylinder. x 51,000. Fig. B.-Median section through the fused basal bodies. x 51,000. Fig. 6.-Oblique section across a basal body. x 63,000. Fig. 7.-Median section through anterior regions of paired cells, IO minutes after mating. Arrow indicates a bubble in the cell walls. x 30,000. A, anterior projection of the fused blepharoplasts; B, basal body; C, central cylinder; D, transverse diaphragm; P, peripheral fibril; W, cell wall. Experimental
Cell Research 15
The flagellar apparatus of Chlamydomonas moewusii
621
Fig. 8. --Median section through anterior regions of paired cells, 10 minutes after mating, showing protoplasmic bridge. x 55,000. Fig. I).-Median section through anterior regions of paired cells, 2 hours after mating. Note that flagellar planes are perpendicular. x 50,000. A, anterior projection of the fused blepharoplasts; B, basal body; BR, protoplasmic bridge; C, central cylinder; V, contractile vacuole; W, cell wall. Experimenial
Cell Research 15
Sarah P. Gibbs, R. A. Lewin and D. E. Philpott The flagella of a series of mutants with impaired motility (see Table I). No abnormal features other than that of length electron microscope. TABLE Mutant
I.
Observations
on the flagella
number
1002 1034, 1038, 1043 1060 1085, 1087 224, 1083, 1084, 1086
Light-microscope Paralysed, Paralysed, Paralysed, Paralysed, “Flagellaless”
of mutants
[2] were also examined were observed with the
of Chlamydomonas
observations
recoverable in dikaryons non-recoverable sterile flagella 2-4 ,U long
moewusii.
E-M observations (2)
Normal Normal (Fig. 2) Normal Otherwise normal Flagella 0.6-1.0 pl long, otherwise normal
When cells of plus and minus mating types of Chlamydomonas moewusii are mixed under suitable conditions, the cells clump and ultimately pair. At first the cells are held together only by adhesion of their flagella, but within a few minutes a protoplasmic bridge is formed between the anterior papillae of the two gametes [l]. Since there is evidence that factors involved in flagellar motility are transferred from one cell to another through the bridge [2], cell pairs were fixed at various intervals from 5 minutes to 4 hours after mating and examined in thin sections. In pairs fixed 5 minutes after mating, no complete bridges were found, but tenuous extensions were occasionally observed projecting from the papillae of the cells. In one case the projections were seen to have met, but had not fused. Cell pairs fixed 10 minutes after mating were seen to be held together by a bridge of homogeneous protoplasm delimited by a well-defined membrane (Fig. 8). In cells mated for 1 to 4 hours, the anterior projections of the blepharoplasts appeared to have grown across the bridge and joined to form an electron-dense, core to the bridge (Fig. 9). In the early stages of mating, bubbles were often found in the anterior region of the cell walls, which apparently break down by vacuolization to permit the formation of the protoplasmic bridge. Summary.-The flagella of Chlamydomonas moewusii are similar to those of other Protista except for a small cylinder which lies within the peripheral fibrils in the basal region of the flagellum and is traversed by a diaphragm. The flagella of the 11 nonmotile mutants examined display no abnormality in internal structure, though in some strains the flagella are only 0.6-1.0 ,u in length. The protoplasmic bridge joining mated cells has an electron-dense core formed by extensions from the anterior projections of the blepharoplasts. REFERENCES 1. 2. 3. 4.
LEWIN, R. A., Biol. Bull. 103, 74 (1952). J. Gen. Microbial. 11, 358 (1954). LEWIN, R. A. and MEINHART, J. O., Can. J. Botany 31, 711 (1953). PHILPOTT, D. E., Expt[. Med. and Surg. 13, 189 (1955).
__
Experimental Cell Research 15
FINE
STRUCTURE
OF THE
FLAGELLAR
CHLAMYDOMONAS SARAH
P. GIBBS,l
Marine
Laboratory,
OF
MOEWUSII
R. A. LEWIN
Biological
APPARATUS
and D. E. PHILPOTT
Woods Hole, Mass., U.S.A.
Received September 10, 1958
AN electron-microscope
examination of the fine structure of the flagellar apparatus of Chlamydomonas moewusii was carried out to support studies on the genetic control of motility in this flagellate [2]. These observations of sectioned material extend previous studies with shadowed whole mounts [3]. Methods.-Cells grown in bacteria-free culture [2] were fixed for 30 minutes in 1 per cent osmium tetroxide buffered with acetate-Verona1 to pH 7.6, washed, dehydrated quickly in an ethanol series, and imbedded in a mixture OF4 parts n-butyl and I part methyl methacrylate. Sections were cut on an experimental microtome [4] and observed with an RCA electron microscope, model EMU-2B. Results.-Each flagellum consists of a cylinder of 9 double peripheral fibrils and a central pair, all imbedded in a structureless matrix and bounded by a membrane, about 100 w thick, continuous with the plasma membrane of the cell (Fig. 1). Each individual fibril appears tubular and is loo-150 A in diameter. The 2 central fibrils project distally about 200 rnp beyond the ends of the peripheral fibrils to form a mucronate tip to the flagellum. Within the cell, the 9 double peripheral fibrils continue to form the basal body of the flagellum. As in most Protista, a cross section through the basal body appears as an electron-dense ring comprising 9 denser areas (Fig. 6). A median longitudinal section of the basal region of the flagellum reveals an H-shaped structure not previously reported in other flagella or cilia (Fig. 4). This consists of an electron-dense cylinder, about 70 rnp in diameter and 120 rnp long, lying within the peripheral fibrils and traversed slightly below its middle by a diaphragm which extends across the flagellum. Fig. 3 shows a cross section through this central cylinder, presumably cut above the transverse diaphragm since the peripheral fibrils are still fairly distinct. The central fibrils appear to end unattached just above the distal end of the cylinder and do not continue into its lumen. The paired flagella arise from opposite sides of an anterior papilla and pass to the anterior through a tunnel in the thickened cell wall. Along the median axis the two blepharoplasts join to share a common lumen (Fig. 5). Extending anteriorly into the papilla from the fused basal bodies is a thick-walled, bluntly-conical projection, whose lumen is at least partly separated from that of the blepharoplasts by one or two transverse partitions (Fig. 5). In some median sections a pair of double membranes, possibly indicating tubules or cisternae, are seen to arise together at the midpoint of the fused blepharoplasts and extend backward, one on each side of the contractile vacuole. No connection with the nucleus could be established. 1 Present address: Biological
Laboratories,
Harvard
University,
Cambridge, Experimental
Mass., U.S.A. Cell Research 15
Sarah P. Gibbs, R. A. Lewin and D. E. Philpoff
Fig. l.-Cross section through a flagellum of a normal cell. x 106,000. Fig. 2.-Cross section through a flagellum of paralysed mutant #1043. x 97,000. Fig. 3.-Cross section through the central cylinder. x 72,000. Fig. 4.-Median section through the central cylinder. x 51,000. Fig. B.-Median section through the fused basal bodies. x 51,000. Fig. 6.-Oblique section across a basal body. x 63,000. Fig. 7.-Median section through anterior regions of paired cells, IO minutes after mating. Arrow indicates a bubble in the cell walls. x 30,000. A, anterior projection of the fused blepharoplasts; B, basal body; C, central cylinder; D, transverse diaphragm; P, peripheral fibril; W, cell wall. Experimental
Cell Research 15
The flagellar apparatus of Chlamydomonas moewusii
621
Fig. 8. --Median section through anterior regions of paired cells, 10 minutes after mating, showing protoplasmic bridge. x 55,000. Fig. I).-Median section through anterior regions of paired cells, 2 hours after mating. Note that flagellar planes are perpendicular. x 50,000. A, anterior projection of the fused blepharoplasts; B, basal body; BR, protoplasmic bridge; C, central cylinder; V, contractile vacuole; W, cell wall. Experimenial
Cell Research 15
Sarah P. Gibbs, R. A. Lewin and D. E. Philpott The flagella of a series of mutants with impaired motility (see Table I). No abnormal features other than that of length electron microscope. TABLE Mutant
I.
Observations
on the flagella
number
1002 1034, 1038, 1043 1060 1085, 1087 224, 1083, 1084, 1086
Light-microscope Paralysed, Paralysed, Paralysed, Paralysed, “Flagellaless”
of mutants
[2] were also examined were observed with the
of Chlamydomonas
observations
recoverable in dikaryons non-recoverable sterile flagella 2-4 ,U long
moewusii.
E-M observations (2)
Normal Normal (Fig. 2) Normal Otherwise normal Flagella 0.6-1.0 pl long, otherwise normal
When cells of plus and minus mating types of Chlamydomonas moewusii are mixed under suitable conditions, the cells clump and ultimately pair. At first the cells are held together only by adhesion of their flagella, but within a few minutes a protoplasmic bridge is formed between the anterior papillae of the two gametes [l]. Since there is evidence that factors involved in flagellar motility are transferred from one cell to another through the bridge [2], cell pairs were fixed at various intervals from 5 minutes to 4 hours after mating and examined in thin sections. In pairs fixed 5 minutes after mating, no complete bridges were found, but tenuous extensions were occasionally observed projecting from the papillae of the cells. In one case the projections were seen to have met, but had not fused. Cell pairs fixed 10 minutes after mating were seen to be held together by a bridge of homogeneous protoplasm delimited by a well-defined membrane (Fig. 8). In cells mated for 1 to 4 hours, the anterior projections of the blepharoplasts appeared to have grown across the bridge and joined to form an electron-dense, core to the bridge (Fig. 9). In the early stages of mating, bubbles were often found in the anterior region of the cell walls, which apparently break down by vacuolization to permit the formation of the protoplasmic bridge. Summary.-The flagella of Chlamydomonas moewusii are similar to those of other Protista except for a small cylinder which lies within the peripheral fibrils in the basal region of the flagellum and is traversed by a diaphragm. The flagella of the 11 nonmotile mutants examined display no abnormality in internal structure, though in some strains the flagella are only 0.6-1.0 ,u in length. The protoplasmic bridge joining mated cells has an electron-dense core formed by extensions from the anterior projections of the blepharoplasts. REFERENCES 1. 2. 3. 4.
LEWIN, R. A., Biol. Bull. 103, 74 (1952). J. Gen. Microbial. 11, 358 (1954). LEWIN, R. A. and MEINHART, J. O., Can. J. Botany 31, 711 (1953). PHILPOTT, D. E., Expt[. Med. and Surg. 13, 189 (1955).
__
Experimental Cell Research 15