A Comparative electron microscopic study of cytoplasmic microtubules and axial unit tubules in a spermatozoon and a protozoan

Cytoplasmic microtubules and axial unit tubules were studied in both sectioned and negatively-stained material. Walls of microtubules of frog lung-fluke (Haematoloechus medioplexus) spermatozoa have a helical substructure, while those of the flagellate, Trypanosoma lewisi, are composed of ten longitudinally-oriented filaments. Cross-bridges occur between some filaments of trypanosome microtubules. Doublet tubules of axial units in both cell types are structurally similar to the trypanosome microtubules, which may indicate similarity of function. Microtubules of fluke spermatozoa appear to be somewhat rigid, are resistant to sonication, and are considered to be mainly supportive. Circular profiles of wall subunits are seen in transverse sections of microtubules of both cell types and in doublet tubules of the trypanosome. Comparisons are made between sectioned and negatively-stained material; while negative-staining better reveals the fundamental substructure of microtubular elements, some distortion appears to occur. In connection with this research, a brief preliminary article demonstrated the presence of subunits in the walls of cytoplasmic microtubules of fluke spermatozoa (Burton, '66). Also, it was shown that the wall of these tubular elements possesses a helical structure, and a diagrammatic representation of the wall structure was set forth.     

Arrangement of tubulin subunits and microtubule-associated proteins in the central-pair microtubule apparatus of squid (Loligo pealei) sperm flagella

This study provides a comprehensive, high-resolution structural analysis of the central-pair microtubule apparatus of sperm flagella. It describes the arrangement of several microtubule-associated "sheath" components and suggests, contrary to previous thinking, that microtubules are structurally asymmetric. The two microtubules of the central pair are different in several respects: the C2 tubule bears a single row of 18-nm-long sheath projections with an axial periodicity of 16 nm, whereas the C1 tubule possesses rows of 9-nm globular sheath components with an axial repeat of 32 nm. The lumen of the C2 tubule always appears completely filled with electron-dense material; that of the C1 tubule is frequently hollow. The C2 tubule also possesses a series of beaded chains arranged around the microtubule; the beaded chains are composed of globular subunits 7.5-10 nm in diameter and appear to function in the pairing of the C1 and C2 tubules. These findings indicate: that the beaded chains are not helical, but assume the form of lock washers arranged with a 16-nm axial periodicity on the microtubule; and that the lattice of tubulin dimers in the C2 tubule is not helically symmetric, but that there are seams between certain pairs of protofilaments. Proposed lattice models predict that, because of these seams, central pair and perhaps all singlet microtubules may contain a ribbon of 2-5 protofilaments that are resistant to solubilization; these models are supported by the results of the accompanying paper (R. W. Linck, and G. L. Langevin. 1981. J. Cell Biol. 89: 323-337.     

Some structural and cytochemical observations on the axial filament complex of lung-fluke spermatozoa

As seen in transverse section, doublet elements of the axial unit of spermatozoa of Haematolocchus medioplexus, a frog lung-fluke, possess walls made up of protofibrillar subunits 50–60 Å in diameter. The partition between A and B members of a doublet element often show extra protofibrils which may partially occlude the “lumen” of the A tubule. Each A tubule possesses outer and inner lateral arms which repeat at longitudinal intervals of about 215 Å and which appear to be structurally dissimilar; the outer arm is expanded at its free end and the inner arm often connects to the B tubule of the adjacent doublet element. Regularly-spaced radial links connect the central sheath of an inner core complex to the A tubules of the peripheral doublet elements. Tests for magnesium-activated ATPase activity provide evidence that the enzyme is associated with the surfaces of doublet elements and the surface of the central sheath. Finally, study of an axial unit which developed in an abnormal manner suggests that normal differentiation of an axial unit may depend on the elaboration of a core complex and radial links.     

Degradation of HMG-CoA reductase-induced membranes in the fission yeast, Schizosaccharomyces pombe

Although the overall structures of flagellar and cytoplasmic microtubules are understood, many details have remained a matter of debate. In particular, studies of the arrangement of tubulin subunits have been hampered by the low contrast of the tubulin subunits. This problem can now be addressed by the kinesin decoration technique. We have shown previously that the recombinant kinesin head domain binds to beta-tubulin, thus enhancing the contrast between alpha- and beta- tubulin in the electron microscope; this allows one to study the arrangement of tubulin dimers. Here we describe the lattices of the four different types of microtubules in eukaryotic flagellar axonemes (outer doublet A and B, central pair C1 and C2). They could all be labeled with kinesin head with an 8-nm axial periodicity (the tubulin dimer repeat), and all of them showed the B-surface lattice. This lattice is characterized by a 0.92-nm stagger between adjacent protofilaments. The B-lattice was observed on the axonemal microtubules as well as on extensions made by polymerizing porcine brain tubulin onto axonemal microtubules in the proximal and distal directions. This emphasizes that axonemal microtubules serve as high fidelity templates for seeding microtubules. The presence of a B-lattice implies that there must be a helical discontinuity ("seam") in the wall. This discontinuity is now placed near protofilaments A1 and A2 of the A- tubule, close to the inner junction between A- and B-microtubules. The two junctions differ in structure: the protofilaments of the inner junction (A1-B10) are staggered roughly by half a dimer, those of the outer junction (A10-B1) are roughly in register. Of the two junctions the inner one appears to have the stronger bonds, whereas the outer one is more labile and opens up easily, generating "composite sheets" with chevron patterns from which the polarity can be deduced (arrow in the plus direction). Decorated microtubules have a clear polarity. We find that all flagellar microtubules have the same polarities. The orientation of the dimers is such that the plus end terminates with a crown of alpha subunits, the minus end terminates with beta subunits which thus could be in contact with gamma-tubulin at the nucleation centers.     

FINE STRUCTURE OF SCIARA COPROPHILA SPERM

Though the fagellum of Sciara sperm arises from a blepharoplast and is characterized by doublet tubules with arms, it differs markedly from the familiar type of flagella in the number and arrangement of its tubules. The axial filament complex in sperm from the testis of Sciara consists of approximately 70 doublet tubules, each with an associated singlet tubule. Near the nucleus these tubules are displaced in an oval array. Posteriorly the oval breaks and coils from one free end so that the axial filament complex at posterior levels has the form of a spiral. The singlet tubules do not extend the full length of the sperm but terminate in order from inside the spiral. Farther posteriorly the axial filament complex reverses the direction of coiling, and the doublets terminate from outside the spiral. Four arms are specifically positioned on the singlet and doublet tubules. A single mitochondrial derivative extends most of the length of the sperm; it consists of a large mass of proteinacious material, a crystalloid located adjacent to the axial filament complex, and peripheral cristae. In the female genital tract, sperm undergo gross morphological changes which include sloughing of practically all the mitochondrial material except the crystalloid, repositioning of the crystalloid, and uncoiling and subsequent recoiling of the axial filament complex into a different configuration. From analysis of serial sections it was determined that the orientation of arms, when the axial filament is viewed from base to tip, is the same as in conventional flagella.     

BRIDGES BETWEEN MICROTUBULES

Bridges between microtubules have been studied with the electron microscope in the axostyle of Saccinobaculus and in various tubule systems of chicken testis, including the helix of tubules surrounding the elongating spermatid nucleus and the flagellum of the sperm tail. In addition to the previously described periodic bridges, evidence is presented that nonperiodic bridges exist between certain tubules. An analysis of axial spacing between adjacent nonperiodic bridges suggests that these structures are attached to periodic binding sites on the microtubule wall, but that not all the binding sites are filled. The bridges appear nonperiodic as a result of random occupancy of some fraction of the periodic sites. The distribution of these binding sites is related to the substructure of the microtubule wall as seen with negative staining and optical diffraction.     

NEW OBSERVATIONS ON FLAGELLAR FINE STRUCTURE : The Relationship Between Matrix Structure and the Microtubule Component of the Axoneme

The sperm flagella of the blowfly Sarcophaga bullata demonstrate the relationship of radial projections in the matrix region to the microtubule organization of the axoneme. The A microtubule of each peripheral doublet is connected to the central sheath by a series of paired radial links. The links lie along the tubule wall with a alternate spacing of about 320/560 A. The distal end of each link is enlarged into a globular head that connects via a transitional link to the helical sheath around the central microtubules. The radial link pairs are disposed in the form of a double helix with a pitch of about 1760 A. It is proposed that a similar organization is common to all cilia and flagella showing ninefold symmetry and must provide, in part, the morphological basis for motility.     

CHLAMYDOMONAS FLAGELLA : I. Isolation and Electrophoretic Analysis of Microtubules, Matrix, Membranes, and Mastigonemes

Methods were developed for the isolation of Chlamydomonas flagella and for their fractionation into membrane, mastigoneme, "matrix," and axoneme components. Each component was studied by electron microscopy and acrylamide gel electrophoresis. Purified membranes retained their tripartite ultrastructure and were shown to contain one high molecular weight protein band on electrophoresis in sodium dodecyl sulfate (SDS)-urea gels. Isolated mastigonemes (hairlike structures which extend laterally from the flagellar membrane in situ) were of uniform size and were constructed of ellipsoidal subunits joined end to end. Electrophoretic analysis of mastigonemes indicated that they contained a single glycoprotein of ~ 170,000 daltons The matrix fraction contained a number of proteins (particularly those of the amorphous material surrounding the microtubules), which became solubilized during membrane removal. Isolated axonemes retained the intact "9 + 2" microtubular structure and could be subfractionated by treatment with heat or detergent. Increasing concentrations of detergent solubilized axonemal microtubules in the following order: one of the two central tubules; the remaining central tubule and the outer wall of the B tubule; the remaining portions of the B tubule; the outer wall of the A tubule; the remainder of the A tubule with the exception of a ribbon of three protofilaments. These three protofilaments appeared to be the "partition" between the lumen of the A and B tubule. Electrophoretic analysis of isolated outer doublets of 9 + 2 flagella of wild-type cells and of "9 + 0" flagella of paralyzed mutants indicated that the outer doublets and central tubules were composed of two microtubule proteins (tubulins 1 and 2) Tubulins 1 and 2 were shown to have apparent molecular weights of 56,000 and 53,000 respectively     

OBSERVATIONS ON SPERMIOGENESIS IN THE FUNGUS GNAT SCIARA COPROPHILA

Although 9-membered centrioles are found in somatic tissues of Sciara, the centriole which lies at the spindle pole of the second meiotic division in male Sciara is composed of a row of approximately 70 short tubules in an oval array. Shortly after telophase of this unequal division, in the daughter cell destined to undergo spermiogenesis, microtubules become confluent with the tubules of the centriole. These tubules have the same density as other cytoplasmic microtubules after glutaraldehyde-OsO₄ fixation and, like them, are not preserved with OsO₄ fixation. As the centriole, now with approximately 70 attached, posteriorly directed, doublet tubules, migrates from the polar to the apolar end of the nucleus to take a final position in an oval groove which forms in the nuclear envelope, the tubules lengthen and become demonstrable after OsO₄ fixation and more electron opaque than other cytoplasmic microtubules following glutaraldehyde-OsO₄ fixation. Later, a singlet tubule appears peripherally to each doublet of the oval and 4 "arms" develop at specific sites on the tubules. Posteriorly, where the oval of tubules becomes discontinuous and forms a spiral, the arrangement of arms is different and the singlet tubules are lacking. Dense solid bodies develop inside this odd flagellum and become enclosed by a smooth double membrane. A single mitochondrial derivative has three components: a central area of homogeneous, moderately electron-opaque, proteinaceous material; a peripheral ring of cristae; and a crystalloid which is specifically oriented with respect to the flagellar tubules.     

THE AXOSTYLE OF SACCINOBACULUS : I. Structure of the Organism and Its Microtubule Bundle

The axostyle of the flagellate Saccinobaculus is a motile ribbon composed of microtubules, cross-bridged to form interconnected rows. We find a centriole-related row of dark-staining tubules near the nucleus at the anterior end of the axostyle. Other tubule rows bind parallel to this primary row, acquire ordered relationships, and become the tubules of the axostyle proper. The number of tubule rows is constant in Saccinobaculus lata from the region near the nucleus to within a few micrometers of the posterior tip of the cell. In Saccinobaculus ambloaxostylus a few tubule rows are added to the axostyle posterior to the nucleus, giving this axostyle a leaf spring construction. The tubules of S. lata are held in rows by links with a 140 Å periodicity along the tubule axis; bridges between rows of tubules are also seen but are not apparently periodic. Each tubule in S. ambloaxostylus shows an axial periodicity of 150 Å due to pairs of arms, one of which is always part of the intrarow link. Interrow bridges in this species run either from tubule to tubule or from tubule to the free arm, but as in S. lata they do not display an obvious axial periodicity. An average unit cell is presented for the axostyle of each species, and the relation of the intertubule links to the microtubule substructure is discussed.     

Different aspects of tubulin polymerization in spermatids of cyprinodontidae (fish,teleost)

Our study concerns 10 genera and 26 species of cyprinodontid fish. In the cytoplasm of spermatids tubulin polymerizes in various forms according to species. We have demonstrated the presence of classic microtubules with a diameter of 24 nm and also of tubules of smaller diameter (15 run) and greater diameter (30 to 50 nm). Microtubules are very numerous in the cytoplasm of certain species. They may be arranged without any order or form bundles which may contain several hundred parallel elements. They never form a manchette. In two species (Aplocheilichthys normani and Epiplatys fasciolatus) only spermatids that degenerate show this peculiarity. The microtubules present two kinds of decorations. The first type are small elements composed of MAP which enable two or more microtubules to link up. The second type are curved tubulin elements that give the microtubule that bear them the appearance of an incomplete doublet. Doublets and triplets may also be formed. Cyprinodontidae spermatocytes and spermatids probably synthesize a very large quantity of tubulin which polymerizes in certain species.     

Sliding of STOP proteins on microtubules

Microtubules are stabilized against cold temperature disassembly by 145-kilodalton proteins [stable tubule only polypeptides (STOPs)] that block the end-wise dissociation of subunits from the polymers. We describe here several kinetic parameters of the interaction of STOPs with microtubules. STOPs will bind to microtubules either during assembly of the polymer or at steady state. The addition appears random on the polymers and does not require the mediation of tubulin subunits. Tubulin subunits compete with microtubules for STOP binding, but binding to the polymers is apparently irreversible. We demonstrate that STOPs do not exchange measurably between polymers at steady state. Nonetheless, a displacement of STOPs within a single polymer is readily demonstrable. We have determined that the displacement is apparently due to a surface translocation, or "sliding", of STOPs on microtubules.     

Evidence for a mixed lattice in microtubules reassembled in vitro

An extensive structural analysis of microtubules assembled in vitro has been carried out using electron microscopy in conjunction with computer analysis based on Fourier transforms and helical diffraction theory. Microtubules assembled in vitro displayed a range of numbers of protofilaments from 12 to 16, with 14 the most abundant (84% in one large sampling). In almost all structures observed protofilaments are staggered to form a characteristic 3-start shallow helix. The presence of the 3-start helix was confirmed by fiber tilting experiments to correct the effects of microtubule flattening. Since α and β tubulin subunits alternate along the protofilaments, continuous helical lattices can be constructed with interactions between adjacent protofilaments involving unlike subunits (type A lattice) or like subunits (type B lattice). However, the 14-protofilament, 3-start microtubules are incompatible with either the A or B-type continuous helical lattice. Evidence is presented which indicates that lattice discontinuities are present which generate features of both the A and B-types, with the latter predominating.     

Configurational changes in helical microtubule frameworks in feeding tentacles of the suctorian ciliateTokophyra

Microtubules at the tip of a resting (non-feeding) tentacle are arranged helically in two concentric tube-shaped arrays. The pitches of the helical paths followed by tubules in the two arrays differ. At the start of feeding these microtubules bend along their longitudinal axes and splay outwards and downwards away from the tentacle tip as it ‘everts’. Tubules in the two arrays slideacross each other as this occurs. Comparison of the fine structure of the tips of feeding and resting tentacles with a dynamic model of the microtubular framework indicates that movement of the tubules is not brought about by active sliding of the tubules against each other or by the action of contractile elements attached along the lengths of tubules. The tips of microtubules forming the inner tube may be pulled downwards by contractile elements in the tentacular pellicle; these tubules apparently push those in. the outer tube to their new positions. The pattern of configurational changes in a tentacle tip at the start of feeding appears to be largely defined by the elastic resistance of the microtubules to bending, and the ways in which tubules are packed and linked together and attached to the pellicle.     

A new interpretation of plasmodesmatal ultrastructure

Summary It is shown that simple, unbranched, plasmodesmata between young xylem ray cells of willow have no direct intercellular continuity apart from the plasmalemma which limits the cytoplasm and lines the plasmodesmatal canal. Each plasmodesma is traversed by a 200 Å diameter tubule (the desmotubule) which has a wall with probably 11 subunits arranged around a central cavity through which runs a 40 Å diameter rod. This rod is connected to the inside of the tubule wall, by fine filaments. At the ends of each plasmodesma the plasmalemma and cell wall are closely appressed to the tubule, thus precluding direct continuity between the cytoplasm of adjacent cells. Through the central part of the plasmodesmata the tubule is separated from the plasmalemma by a 90–100 Å wide gap. Cytoplasmic microtubules in the same tissue have a diameter of approximately 250 Å and a wall probably composed of 13 subunits: both desmotubules and cytoplasmic microtubules therefore have a centre-to-centre subunit spacing of about 47 Å. It is suggested that the desmotubules are not microtubules but may be nuclear spindle fibres which become trapped in the wall during cell plate formation. The endoplasmic reticulum, while closely approaching the plasmodesmata, is not continuous across them. It is thought most unlikely that the endoplasmic reticulum traverses plasmodesmata, as the dimensions of the central tubule — found here as well as by other workers — are smaller than those which would be expected to allow a stable molecular configuration in a unit membrane. The plasmalemma, where it lines the plasmodesmatal canal, appears to have particulate subunits in the outer opaque layers and the presence of these subunits may be attributable to the need for stability in membranes arranged about so small a radius.     

Microtubule organization in germinated pollen of the conifer Picea abies (Norway spruce,Pinaceae)

The organization of microtubules in germinated pollen of the conifer Picea abies (Norway spruce, Pinaceae) was examined using primarily confocal microscopy. Pollination in conifers differs from angiosperms in the number of mitotic divisions between the microspore and the sperm and in the growth rate of the pollen tube. These differences may be orchestrated by the cytoskeleton, and this study finds that there are important functional differences in microtubule organization within conifer pollen compared to the angiosperm model systems. Pollen from P. abies contains two degenerated prothallial cells, a body cell, a stalk cell, and a vegetative cell. The body cell produces the sperm. In the vegetative cell, microtubules form a continuous network from within the pollen grain, out through the aperture, and down the length of the tube to the elongating tip. Within the grain, this network extends from the pollen grain wall to the body and stalk cell complex. Microtubules within the body and stalk cells form a densely packed array that enmeshes amyloplasts and the nucleus. Microtubule bundles can be traced between the body and stalk cells from the cytoplasm of the body cell to the adjoining cell wall and into the cytoplasm of the stalk cell. Body and stalk cells are connected by plasmodesmata. The organization of microtubules and the presence of plasmodesmata suggest that microtubules form a path for intercellular communication by projecting from the cytoplasm to interconnecting plasmodesmata. Microtubules in the elongating tube form a net axial array that ensheathes the vegetative nucleus. Microtubules are enriched at the elongating tip, where they form an array beneath the plasma membrane that is perpendicular to the direction of tube growth. This enriched region extends back 20 μm from the tip. There is an abrupt transition from a net perpendicular to a net axial organization at the edge of the enriched region. In medial sections, microtubules are present in the core of the elongating tip. The organization of microtubules in the tip differs from that seen in angiosperm pollen tubes.     

Tubulin protofilaments and kinesin-dependent motility

Microtubules are built of tubulin subunits assembled into hollow cylinders which consist of parallel protofilaments. Thus, motor molecules interacting with a microtubule could do so either with one or several tubulin subunits. This makes it difficult to determine the structural requirements for the interaction. One way to approach the problem is to alter the surface lattice. This can be done in several ways. Proto-filaments can be exposed on their inside (C-tubules or "sheets"), they can be made antiparallel (zinc sheets), or they can be rolled up (duplex tubules). We have exploited this polymorphism to study how the motor protein kinesin attached to a glass surface interacts and moves the various tubulin assemblies. Microtubules glide over the surface along straight paths and with uniform velocities. In the case of C-tubules, approximately 40% glide similarly to microtubules, but a major fraction do not glide at all. This indicates (a) that a full cylindrical closure is not necessary for movement, and (b) that the inside surface of microtubules does not support gliding. With zinc sheets, up to 70% of the polymers move, but the movement is discontinuous, has a reduced speed, and follows along a curved path. Since zinc sheets have protofilaments alternating in orientation and polarity, this result suggests that in principle a single protofilament can produce movement, even when its neighbors cannot. Duplex microtubules do not move because they are covered with protofilaments coiled inside out, thus preventing the interaction with kinesin. The data can be explained by assuming that the outside of one protofilament represents the minimal track for kinesin, but smooth gliding requires several parallel protofilaments. Finally, we followed the motion of kinesin-coated microbeads on sea-urchin sperm flagella, from the flagellar outer doublet microtubules to the singlet microtubule tips extending from the A-tubules. No change in behavior was detected during the transition. This indicates that even if these microtubules differ in surface lattice, this does not affect the motility.     

ELECTRON MICROSCOPE STUDIES OF SPERMATOZOA OF RHYNCHOSCIARA SP : I. Disruption of Microtubules by Various Treatments

The flagellar complex of the unusual motile spermatozoon of the fungus gnat, Rhynchosciara sp, does not conform to the usual "9 + 2" filament pattern but rather consists of over 350 pairs of filaments (doublet microtubules) distributed in a spiral array. Experiments were designed to disrupt and extract flagellar microtubular components from spermatozoa of the fungus gnat. Pepsin, chymotrypsin, potassium iodide, urea, and heat were used to extract specific portions of microtubule walls Such experiments provide information on the composition of the wall and the existence of wall sites selectively sensitive to various treatments Results obtained include: (a) doublet microtubules are comprised at least in part of protein, and all subunits are probably not identical; (b) a portion of the B subfiber is apparently more sensitive to disruption than other portions of the doublet microtubule; and (c) the ac cessory singlet microtubules may be chemically different from the doublet microtubules     

Flagellar doublet microtubules: fractionation of minor components and alpha-tubulin from specific regions of the A-tubule.

Proteins occurring minor amounts with purified sperm flagellar doublet microtubules were identified and studied by SDS-gel electrophoresis. Methods were developed to solubilize selectively these minor components; electron microscopy (EM) of the fractionated products revealed possible locations of these proteins in the tubule. Doublet microtubules were prepared from sea-urchin (Echinus esculentus and Stronglyocentrotus droebachiensis) and scallop (Pecten maximus) sperm by dialysing flagellar axonemes against 2 mM Tris-0-2 mM EDTA-0-5 mM DTT. EM indicates that these doublet tubule preparations retain at least 70% of their radial spokes; cross-sections show a globule or fibre applied to the inside wall of the A-tubule, across from the inner B-tubule junction. On SDS-gels these preparations separate into at least 10 minor bands, accounting for 20-30% of the total protein; the remaining 75 +/- 4% migrates as tubulin. For E. esculentus the molecular weights and relative amounts of these components are: Component Ee 8 (150000 Daltons; 1%), 11 (114000; 2-5%), 15 (89000; 2%), 16 (80000; 2-5%), 17 (74000; 2%), 18 (69000; 2%), 19 (66000; 2%), 21 (48000; 4-5%), 22 (45000; 3%) and 23 (44500; 3%). Treatment of sea-urchin tubules with 0-1-0-5% sarkosyl, 0-1-0-3 M KSCN or 0-3-0-6 M KI results in the selective solubilization of: first, component 8 and some B-subfibre tubulin; second, components 11 and 23 and the remaining B-subfire tubulin; third, most of the A-subfire tubulin and components 17, 18 and 19. Thermal fractionation extracts none of these components, suggesting they are principally associated with the A-tubule. Finally 25-35% of the original protein is resistant to solubilization, and appears in the EM as ribbons of 3 protofilaments with 16-nm axial repeats. The resistant ribbons contain components 15, 16, 21 and 22 (plus component 20 in S. droebachiensis) in addition to 25 +/- 4% of the total tubulin. The data support the existence of two stable moieties in each doublet tubule: (1) a ribbon of 3 protofilaments and (2) either a second ribbon of 3 protofilaments or an equivalent amount of tubulin in some other form. EM images suggest that one ribbon forms the lateral side of the A-tubule (e.g. protofilaments A1,2,3 or A13,1,2 in the model) and that the globule applied to A13 may be a multisubunit complex of remaining minor components. Treatment of scallop tubules with 0-3 M KSCN preferentially extracts alpha-tubulin, yielding ribbons 1-4 protofilaments wide. The significance of this finding is discussed.     

ELECTRON MICROSCOPIC OBSERVATIONS ON TUBULAR STRUCTURES INVOLVED IN CRYSTAL FORMATION IN THE RED ALGA PLEONOSPORIUM SQUARRULOSUM (HARV.) ABBOTT1

Hexagonal or angular crystalline inclusions in Pleonosporium (Naeg.) Hauck vegetative cells were examined using electron microscopy. Ultrastructural analysis reveals that the inclusions initially contain tubular elements resembling microtubules but, with continued differentiation, are transformed into rod containing crystals. The tubular structures initially measure 25 nm in diameter. Scattered tubules become arranged in a parallel and alternate pattern and undergo subsequent enlargement to approximately 29 nm. Following enlargement, each tubule apparently disaggregates into rods that form a crystal having hexagonally arranged rod-like subunits. It is suggested that these tubules may represent microtubules and the resultant crystals are composed of tubulin.