EXPRESSION OF PRIMARY CILIA IN MAMMALIAN CELLS

Mammalian cellsin vivofrequently express primary cilia. Although some fully differentiated cell types rarely, if ever, express them, most do, indicating that they are regular cell organelles. Their expression can also be exploredin vitro, where conditions—physical and chemical, intrinsic and extrinsic—permit experimental approaches which give far greater control thanin vivo. This ‘state of the art’ paper covers briefly the general biology of primary cilia, highlights the current situation with regard to our understanding of their relevance and importance in cell biology from various facets of our recent research, much of it in collaboration with other laboratories world-wide, and outline future work aimed at answering some basic and applied questions about them, within a context of an increasing awareness that signalling between cells is of the utmost importance in understanding proliferation control and its value in cancer research, the major remit of this unit.     

THE REGENERATION OF CILIA IN PARTIALLY DECILIATED TETRAHYMENA

Partial deciliation of Tetrahymena resulted in cells losing 75% of their cilia, with the balance being paralyzed. The paralyzed cilia are resorbed in the first 20 min after partial deciliation, and regeneration of cilia begins before resorption is completed. Inhibition of protein synthesis with cycloheximide does not inhibit ciliary resorption or regeneration, whereas vinblastine sulfate inhibits regeneration but not resorption. Inhibition of regeneration occurs in completely deciliated cells when they are treated with cyclohexmimide or vinblastine sulfate. It is concluded that the resorbing cilia contribute materials which allow regeneration to occur in the absence of protein synthesis. The volume of cilia regenerated in the presence of cycloheximide in partially deciliated cells is greater than the ciliary volume which is resorbed. This suggests the Tetrahymena cells have a pool of ciliary precursors. This pool does not contribute materials for regeneration in completely deciliated cells which are treated with cycloheximide. It is concluded that resorbing cilia in partially deciliated cells contribute materials which potentiate assembly of cilia from the pool of precursors.     

EVOLUTION OF DEVELOPMENTAL PROGRAMS IN VOLVOX (CHLOROPHYTA)1

The volvocine green algal genus Volvox includes ～20 species with diverse sizes (in terms of both diameter and cell number), morphologies, and developmental programs. Two suites of characters are shared among distantly related lineages within Volvox. The traits characteristic of all species of Volvox—large (>500) numbers of small somatic cells, much smaller numbers of reproductive cells, and oogamy in sexual reproduction—have three or possibly four separate origins. In addition, some species have evolved a suite of developmental characters that differs from the ancestral developmental program. Most multicellular volvocine algae, including some species of Volvox, share an unusual pattern of cell division known as palintomy or multiple fission. Asexual reproductive cells (gonidia) grow up to many times their initial size and then divide several times in rapid succession, with little or no growth between divisions. Three separate Volvox lineages have evolved a reduced form of palintomy in which reproductive cells are small and grow between cell divisions. In each case, these changes are accompanied by a reduction in the rate of cell division and by a requirement of light for cell division to occur. Thus, two suites of characters—those characteristic of all Volvox species and those related to reduced palintomy—have each evolved convergently or in parallel in lineages that diverged at least 175 million years ago (mya).     

OLFACTORY CILIA IN THE FROG

Olfactory epithelium from the frog was examined in the living state by light microscopy and in the fixed state by electron microscopy. Particular attention was paid to the layer of cilia and mucus which covers the surface of the epithelium. The olfactory cilia differed from typical cilia in that they (a) arose from bipolar neurons and had centrioles near their basal bodies, (b) were up to 200 microns in length, of which the greater part was a distal segment containing an atypical array of ciliary fibers, (c) were often immotile, (d) had their distal segments arranged in parallel rows near the surface of the mucus, and (e) had many vesicles along their shafts and had splits in the array of fibers in their distal segments. These specializations make the olfactory cilia similar to cilia found on other sensory cells and support the theory that they are the locus where electrical excitation in the olfactory organ is initiated by contact with odorous substances.     

THE GENETIC BASIS OF MALE POTENCY IN VOLVOX CARTERI F. NAGARIENSIS (CHLOROPHYCEAE)1,2

Sexual males of Volvox carteri Stein f. nagariensis Iyengar secrete a hormone which induces sexual rather than asexual development in the next generation. Male recombinants from the original strains isolated from Japan showed an increase in potency of up to 100 times that of the original male clone, with later descendants showing potencies ranging from 100 times to 1/1000 the potency of this male. Potency is defined as the extent to which the hormone produced in 1 ml of medium by one parental spheroid containing 16 male spheroids can be diluted and retain its capacity to induce sexual differentiation. This is recorded as the percent of female response in the tube of limiting dilution, asexual females of the normal female strain being used as assay inoculum in a 1 in 10 dilution series of samples of male fluid containing the sexual hormone. Investigation of the genetic basis for potency in this species included selection of high and low potency male clones and crossing with sib females or backcrossing with original female. Results of the various crosses are interpreted as multigenic system not linked with sex, with more than one genotypic class per dilution level and with more than one genotypic class per apparent phenotypic class. Possible genic interactions are described in their relevance to the biosynthesis of a glycoprotein molecule, the sexual hormone having been characterized as a glycoprotein by Starr and Jaenicke (18).     

INVERSION IN VOLVOX (CHLOROPHYCEAE)1

The asexual embryos of Volvox turn themselves inside out (invert) during development. Data presented indicate that inversion of the embryos is the result of several simple cellular shape changes, coordinated in space and time. Using whole embryos, cell groups and individual embryonic cells isolated by watchmaker's forceps and pressure on the coverslip, it was shown that the phialopore (opening) enlargement and concurrent stretching of the border cells is due to a constriction formed at the equator. However, if the posterior hemisphere is removed, this constriction no longer effects the expansion of the phialopore (which is in the anterior hemisphere) because the equatorial region is no longer anchored and has no base to support the outwardly directed force against the phialopore cells. If the posterior hemisphere is isolated several hours before inversion, the opening resulting from the incision acts as a “phialopore” and the direction of inversion is reversed. Individual cells and cells in groups undergo the same shape changes as corresponding cells in an intact embryo during inversion. This suggests that these cellular deformations are autonomous and inversion is a result of them.     

FINE STRUCTURE OF THE CILIA OF ROTIFERS

The fine structure of the coronal cilia of the rotifer Philodina citrina has been studied in detail. Specimens were fixed with OsO₄ and embedded in butyl—methyl methacrylate, Epon 812, or Vestopal and sectioned with a Porter-Blum microtome. The details of structure of the rootlets, basal bodies, basal plates, and free cilia are described. The general structure of the rotifer ciliary apparatus conforms well to that established for other species. One of the main observations is the difference in structure of the peripheral filaments in the opposing halves of a cross-section of the free cilium. Also, in longitudinal sections evidence is offered for the existence of a helical structure in the peripheral filaments.     

THE STRUCTURE AND FORMATION OF CILIA AND FILAMENTS IN RUMEN PROTOZOA

The large oligotrich rumen protozoa Diplodinium ecaudatum and Ophryoscolex caudatus have been studied by electron microscopy during interphase and division. The structure of mature cilia is contrasted with that seen during their formation particularly in a tuft where development lags and is arrested. Here the shaft is only a few micra long and is composed of filaments that have circular cross-sections not in the typical circular arrangement. In their diameter and appearance the filaments are similar to filaments associated with the nuclei during division. The macronucleus has within it randomly directed filaments, while the micronucleus contains well aligned filaments and other arrangements typical of an intranuclear mitotic process. An extranuclear filament system is also present and is elaborated during division. The infraciliary filament system is particularly elaborate in these organisms. Filaments ranging from 14 to 22 mµ have been observed with some tendency for a bimodal distribution in diameters of 15 and 21 mµ. Formation of such filaments has been observed and consists of an initial orientation of very fine elements followed by filament formation. The observations are discussed in relation to filament involvements in cell movements. The concepts are discussed that filaments are metastable structures and that the transitions from one state to another are functionally significant.     

光捕捉活细胞染色体

本文综合报道了作者近数年来以PTK_2细胞为实验材料,用Nd:YAG激光器所发射的1.06微米波长和氩离子泵浦Titanium-Sapphire激光所发射的700—760毫微米波长的连续激光微光束作为光捕捉在显微操作染色体方面的一些主要实验结果。所得结果表明光捕捉可诱发中期细胞的落后染色体向中期板加速移动,抓住后期细胞的一对染色体,使其停留在中期板保持静止不动,而其余的染色体对照常进行染色单体的分离並移向两极,在后期一直用光捕捉抓住的那对染色单体,最终在胞质分裂时将进入一个子细胞,或丢失在分裂沟中或两染色单体分开,各自分别进入原相对的子细胞。作为光捕捉Titanium-Sapphire激光器发射的700—760毫微米波长的激光束,比Nd:YAG激光的1.06微米波长能在更高的输出能量水平下操作而产生较小的对细胞损伤的副作用,从而更容易操作染色体。在适宜的输出能量水平下操作,光捕捉不会对细胞造成损伤,受光捕捉的细胞一般都能继续分裂直至形成两个子细胞。实验结果证明光捕捉技术是一项研究活细胞纺锤体、染色体运动等细胞生物学问题而又不损伤细胞的良好工具。光捕捉技术也可能对诱发单体、三体细胞,研究细胞遗传提供新的手段。     

DEMONSTRATION OF CRYPTIC INCOMPATIBILITY IN DISTYLOUS AMSINCKIA DOUGLASIANA

Hand pollinations experiments with distylous Amsinckia douglasiana A. DC. (Boraginaceae) revealed that legitimate (intermorph) crosses produce more seed than self-pollinations and that self-pollinations yield more seed than illegitimate (intramorph) crosses. We tested the occurrence of cryptic incompatibility in both style-length morphs by pollinating them with a mixture of legitimate and illegitimate pollen, each homozygous for a different electrophoretically distinguishable allele of phosphoglucose isomerase. The success of intramorph pollen was greatly reduced in mixture; only about 4% of the offspring of both pins and thrums were sired by illegitimate pollen. One pin and one thrum had sufficiently high seed set that the results cannot be attributed to selective abortion of embryos. In those individuals, discrimination against illegitimate pollen must have occurred on the stigma or in the style. Based on our findings, it appears that illegitimate matings are rare in natural populations. The progeny of legitimate crosses was biased toward pins, which suggests some regulation of morph ratios independent of the style-length locus. Prezygotic discrimination against the thrum genotype or selective abortion of embryos with the thrum genotype are both possible, but selective mortality of seedlings with the thrum genotype cannot be ruled out for some families.     

SOMATIC CELL FLAGELLAR APPARATUSES IN TWO SPECIES OF VOLVOX (CHLOROPHYCEAE)1

The somatic cell flagellar apparatuses of Volvox carteri f. weismannia (Powers) Iyengar and V. rousseletii G. S. West have parallel or nearly parallel basal bodies which are separated at their proximal ends. The four microtubular rootlets alternate between two and four members, and all are associated with a striated microtubular associated component (SMAC) that runs between the basal bodies. In addition, each half of the flagellar apparatus apparently rotates during development and loses the 180° rotational symmetry characteristic of most unicellular chlorophycean motile cells. All of these features appear necessary for efficient motion of a colony composed of numerous radially arranged cells. However, the structural details of the flagellar apparatuses of these two species differ. The distance between flagella is greater in V. rousseletii than in V. carteri. One distal striated fiber and two proximal striated fibers connect the basal bodies in V. carteri, but both types of fibers are absent from V. rousseletii. In the latter species, a striated fiber wraps around each of the basal bodies and attaches to the rootlets and the SMAC. No such fiber is present in V. carteri. Since the similarities in the flagellar apparatuses can be explained as a result of adaptation for efficient colonial motion in organisms with similar colonial morphology, the differences suggest a wider phylogenetic distance than previously believed.