bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel cytoplasmic dynein heavy chain: expression of DHC1b in mammalian ciliated epithelial cells
Authors: Criswell Peggy S. , Ostrowski Lawrence E. , Asai David J.
Published: April 2021

Organisms that have cilia or flagella express over a dozen dynein heavy chain genes. Of these heavy chain genes, most appear to encode axonemal dyneins, one encodes conventional cytoplasmic dynein (MAP1C or DHC1a), and one, here referred to as DHC1b, encodes an unclassified heavy chain. Previous analysis of sea urchin DHC1b (Gibbons et al. (1994)Mol. Biol. Cell 5, 57-70) indicated that this isoform is either an axonemal dynein with an unusual protein sequence or a cytoplasmic dynein whose expression increases during ciliogenesis. In the present study, we examined the expression of DHC1b in rat tissues. The DHC1b gene is expressed in all tissues examined, including unciliated liver and heart cells. In contrast, rat axonemal dyneins are only expressed in tissues that produce cilia or flagella. In cultured rat tracheal epithelial (RTE) cells, DHC1b is expressed in undifferentiated cells and increases in expression during ciliogenesis. In contrast, the expression of conventional cytoplasmic dynein, DHC1a, does not change during RTE differentiation and axonemal dynein is not expressed until after differentiation commences. In order to examine the expression of DHC1b protein, we produced an isoform-specific antibody to a synthetic peptide derived from the rat DHC1b sequence. The antibody demonstrated that DHC1b is a relatively minor component of partially purified cytoplasmic dynein. Indirect immunofluorescence microscopy revealed that DHC1b is not detected in cilia and remains in the cytoplasm of ciliated RTE cells, often accumulating at the apical ends of the cells. These results suggest that DHC1b is a cytoplasmic dynein that may participate in intracellular trafficking in polarized cells.
Regulation of flagellar dynein by an axonemal type-1 phosphatase in Chlamydomonas
Authors: Habermacher Geoffrey , Sale Winfield S.
Published: April 2021

Physiological studies have demonstrated that flagellar radial spokes regulate inner arm dynein activity in Chlamydomonas and that an axonemal cAMP-dependent kinase inhibits dynein activity in radial spoke defective axonemes. These studies also suggested that an axonemal protein phosphatase is required for activation of flagellar dynein. We tested whether inhibitors of protein phosphatases would prevent activation of dynein by the kinase inhibitor PKI in Chlamydomonas axonemes lacking radial spokes. As predicted, preincubation of spoke defective axonemes (pf14 and pf17) with ATPγS maintained the slow dynein-driven microtubule sliding characteristic of paralyzed axonemes lacking spokes, and blocked activation of dynein-driven microtubule sliding by subsequent addition of PKI. Preincubation of spoke defective axonemes with the phosphatase inhibitors okadaic acid, microcystin-LR or inhibitor-2 also potently blocked PKI-induced activation of microtubule sliding velocity: the non-inhibitory okadaic acid analog, 1-norokadaone, did not. ATPγS or the phosphatase inhibitors blocked activation of dynein in a double mutant lacking the radial spokes and the outer dynein arms (pf14pf28). We concluded that the axoneme contains a type-1 phosphatase required for activation of inner arm dynein.

We postulated that the radial spokes regulate dynein through the activity of the type-1 protein phosphatase. To test this, we performed in vitro reconstitution experiments using inner arm dynein from the double mutant pf14pf28 and dynein-depleted axonemes containing wild-type radial spokes (pf28). As described previously, microtubule sliding velocity was increased from ∼2 μm/second to ∼7 μm/second when inner arm dynein from pf14pf28 axonemes was reconstituted with axonemes containing wild-type spokes. In contrast, pretreatment of inner arm dynein from pf14pf28 axonemes with ATPγS, or reconstitution in the presence of microcystin-LR, blocked increased velocity following reconstitution, despite the presence of wild-type radial spokes. We conclude that the radial spokes, through the activity of an axonemal type-1 phosphatase, activate inner arm dynein by dephosphorylation of a critical dynein component. Wild-type radial spokes also operate to inhibit the axonemal cAMP-dependent kinase, which would otherwise inhibit axonemal dynein and motility.
Identification of endoplasmic reticulum in the primitive eukaryote Giardia lamblia using cryoelectron microscopy and antibody to BiP
Authors: Soltys Bohdan J. , Falah Mizied , Gupta Radhey S.
Published: April 2021

Giardia lamblia trophozoites contain a complex endomembrane system as demonstrated by fluorescence and cryoelectron microscopy. The endomembrane system was weakly detected in live cells using the fluorescent membrane dye 3,3′-dihexyloxacarbocyanine iodide. The definitive identification of endoplasmic reticulum required the development of a molecular label. We expressed Giardial Bip in Escherichia coli and raised a polyclonal antibody to the purified protein. In western blots, the antibody was specific for Giardial Bip and did not react with human, monkey and rodent homologs. By immunofluorescence microscopy in methanol fixed cells the antibody visualized tubular structures and other subcellular components that required characterization by electron microscopy. Using cryotechniques we directly demonstrate the presence of a complex endomembrane system at the ultrastructural level. In conjunction with Bip immunogold labeling of cryosections we identify: (1) endoplasmic reticulum cisternae and tubules; (2) stacked perinuclear membranes; and (3) Bip presence in the nuclear envelope. Both the endoplasmic reticulum and nuclear envelope were found either with or without a cleft region suggesting each may contain common specialized sub-regions. In stacked perinuclear membranes, which may represent either multilamellar endoplasmic reticulum or a Golgi apparatus, Bip labeling was restricted to peripheral layers, also suggesting specialized sub-regions. Labeled endomembrane systems could be observed associated with microtubule structures, including axonemes and the adhesive disk. The presence of an extensive endomembrane system in Giardia lamblia, which represents one of the earliest diverging eukaryotic species, supports the view that both the nucleus and endomembrane system co-evolved in a common ancestor of eukaryotic cells.
Function of spindle microtubules in directing cortical movement and actin filament organization in dividing cultured cells
Authors: Fishkind Douglas J. , Silverman John D. , Wang Yu-li
Published: April 2021

The mitotic spindle has long been recognized to play an essential role in determining the position of the cleavage furrow during cell division, however little is known about the mechanisms involved in this process. One attractive hypothesis is that signals from the spindle may function to induce reorganization of cortical structures and transport of actin filaments to the equator during cytokinesis. While an important idea, few experiments have directly tested this model. In the present study, we have used a variety of experimental approaches to identify microtubuledependent effects on key cortical events during normal cell cleavage, including cortical flow, reorientation of actin filaments, and formation of the contractile apparatus. Single-particle tracking experiments showed that the microtubule disrupting drug nocodazole induces an inhibition of the movements of cell surface receptors following anaphase onset, while the microtubule stabilizing drug taxol causes profound changes in the overall pattern of receptor movements. These effects were accompanied by a related set of changes in the organization of the actin cytoskeleton. In nocodazole-treated cells, the three-dimensional organization of cortical actin filaments appeared less ordered than in controls. Measurements with fluorescencedetected linear dichroism indicated a decrease in the alignment of filaments along the spindle axis. In contrast, actin filaments in taxol-treated cells showed an increased alignment along the equator on both the ventral and dorsal cortical surfaces, mirroring the redistribution pattern of surface receptors. Together, these experiments show that spindle microtubules are involved in directing bipolar flow of surface receptors and reorganization of actin filaments during cell division, thus acting as a stimulus for positioning cortical cytoskeletal components and organizing the contractile apparatus of dividing tissue culture cells.
Determination of spatial patterns of DNA damage and repair in intestinal crypts by multi-cell gel electrophoresis
Authors: Brooks Roger A. , Winton Douglas J.
Published: April 2021

We have developed a method to quantitate DNA strand breaks as a measure of DNA damage and repair in intact, isolated intestinal crypts. The assay is a modified form of the single-cell gel electrophoresis or ‘comet’ assay. By maintaining the spatial relationship between the cells we were able to characterise the repair response and the susceptibility to DNA damage of cells as a function of their position in the crypt. All cells were equally repair competent over the first 30 minutes of the repair of UV-C and γ-radiation induced lesions. DNA damage was equally distributed following γ-radiation but following incubation with the topoisomerase II inhibitor etoposide, damage was greater in the lower crypt with an unusual component to the comet tail which was tapered, implying an incremental change in susceptibility by cell position. This tapered component of the comet tail resolved rapidly after removal of etoposide. The pattern of damage produced by hydrogen peroxide was dose dependent with lower doses producing more strand breaks in the base of the crypt-an effect lost at higher doses. The assay has the ability to detect differences between cells in their susceptibility to DNA damage and their subsequent repair response which may vary with their proliferative or differentiative status.
Tenascin-X expression in tumor cells and fibroblasts: glucocorticoids as negative regulators in fibroblasts
Authors: Sakai Takao , Furukawa Yusuke , Chiquet-Ehrismann Ruth , Nakamura Mitsuru , Kitagawa Seiichi , Ikemura Toshimichi , Matsumoto Ken-ichi
Published: April 2021

Tenascin-X has recently been shown to be a novel member of the tenascin family and its distribution is often reciprocal to that of tenascin-C in the developing mouse embryo. We have investigated the expression of tenascin-X in fibroblasts and carcinoma cells in culture. Tenascin-X protein was secreted in vitro in the conditioned media at an apparent molecular mass of ∼450 kDa. In addition fibroblasts contained a major tenascin-X isoform of 220 kDa. On northern blots, a single major transcript with a size of ∼13 kb was detected. No overexpression of tenascin-X protein was found in primary fibroblasts of the tenascin-C-gene knockout mice. Steroid hormone glucocorticoids, were found to downregulate tenascin-X mRNA levels and protein synthesis in fibroblasts but not carcinoma cells at physiological concentrations. None of the growth factors or cytokines examined affected the expression level of tenascin-X. As in vivo study, carcinoma cells were transplanted into nude mice. In contrast to the ubiquitous presence of tenascin-X in adult skin, expression of tenascin-X protein during tumorigenesis was found to be downregulated considerably not only in tumor cells themselves but also in tumor stroma. These findings provide evidence that the expression of tenascin-X can be influenced by stromal-epithelial interactions. We have identified glucocorticoids as physiological inhibitors of tenascin-X and suggest that glucocorticoids may in part participate in the downregulation of tenascin-X in fibroblasts in vivo.
Cytokinesis in Aspergillus nidulans is controlled by cell size, nuclear positioning and mitosis
Authors: Wolkow Tom D. , Harris Steven D. , Hamer John E.
Published: April 2021

The mycelium of Aspergillus nidulans is composed of multinucleate cellular compartments delimited by crosswalls called septa. Septum formation is dependent on mitosis and requires the recruitment of actin to the site of septum formation. Employing a collection of temperature sensitive nuclear distribution (nudA2, nudC3 and nudF7), nuclear division (nimA5, hfaB3), and septation (sepD5, sepGl) mutants, we have investigated the interdependency among nuclear positioning, mitosis, and cell growth in structuring the cellular compartments of A. nidulans. The cellular compartments of nud+ strains were highly uniform with regard to nuclear distribution and averaged 38 µm in length. Incubation of nud mutants at semi-restrictive temperature resulted in aberrant nuclear distribution that appeared to direct the formation of variable-sized cellular compart ments, ranging from 5 µm to greater than 81 µm. In germinating spores, the first septum forms at the basal end of the germ tube following the third round of nuclear division. Germlings must undergo mitosis in order to form a septum. Temperature-sensitive mitotic mutants were used to show that a single nuclear division is sufficient to activate septum formation, provided a critical cell size has been attained. In mitotic mutants and wild-type cells, delays in nuclear division resulted in the misplacement of the first septum. These results strongly support the role of mitotic nuclei in determining septal placement, and suggest that cell size control is post-mitotic in A. nidulans.
TGFβl promotes actin cytoskeleton reorganization and migratory phenotype in epithelial tracheal cells in primary culture
Authors: Boland Sonja , Boisvieux-Ulrich Emmanuelle , Houcine Odile , Baeza-Squiban Armelle , Pouchelet Marcel , Schoëvaërt Damien , Marano Francelyne
Published: April 2021

In the present study we have investigated the effects of transforming growth factor β (TGFβ1) on rabbit tracheal epithelial cells in primary culture, with respect to cell proliferation and differentiation. Epithelial tracheal cells derived from an explant plated on an extracellular matrix, formed an outgrowth resulting from cell division and cell migration. TGFβ1 treatment produced a negative effect on cell proliferation, but in contrast, promoted a marked enhancement of cell migration and increase in outgrowth surface. TGFβ1 induced marked cell shape changes, including cell spreading and lack of stratification, associated with reduced cell-cell contacts and increased cell-substratum anchorage, as seen by electron microscopic observations. Immunocytological studies demonstrated major TGFβ1-induced actin cytoskeleton reorganization, corresponding to the development of a basal stress fiber network and decrease of the annular cell border, without affecting the tight junctions. The migratory phenotype was approached by microcinematography which clearly showed that TGFβ1 triggered a stimulatory effect on migration of epithelial cells, determined using an image analyzing system. Present findings suggest a beneficial role for TGFβ1 during wound healing in providing the acquisition of a migratory phenotype, with a higher capacity to migrate either on collagen or on different extracellular matrix components including laminin and fibronectin. Conversely, present data are not consistent with a squamous response to TGFβ1, since metaplastic differentiation did not occur, as characterized by cytokeratin expression and cross-linked envelopes formation.
Chromatin organization in the homogeneously staining regions of a methotrexate-resistant mouse cell line: Interspersion of inactive and active chromatin domains distinguished by acetylation of histone H4
Authors: Nicol Linda , Jeppesen Peter
Published: April 2021

We have analyzed the organization of the homogeneously staining regions (HSRs) in chromosomes from a methotrexate-resistant mouse melanoma cell line. Fluorescence in situ hybridization techniques were used to localize satellite DNA sequences and the amplified copies of the dihydrofolate reductase (DHFR) gene that confer drug-resistance, in combination with immunofluorescence using antibody probes to differentiate chromatin structure. We show that the major DNA species contained in the HSRs is mouse major satellite, confirming previous reports, and that this is interspersed with DHFR DNA in an alternating tandem array that can be resolved at the cytological level. Mouse minor satellite DNA, which is normally located at centromeres, is also distributed along the HSRs, but does not appear to interfere with centromere function. The blocks of major satellite DNA are coincident with chromatin domains that are labelled by an autoantibody that recognizes a mammalian homologue of Drosophila heterochromatin-associated protein 1, shown previously to be confined to centric heterochromatin in mouse. An antiserum that specifically recognizes acetylated histone H4, a marker for active chromatin, fails to bind to the satellite DNA domains, but labels the intervening segments containing DHFR DNA. We can find no evidence for the spreading of the inactive chromatin domains into adjacent active chromatin, even after extended passaging of cells in the absence of methotrexate selection.
The spacer protein Spc110p targets calmodulin to the central plaque of the yeast spindle pole body
Authors: Spang Anne , Grein Katrin , Schiebel Elmar
Published: April 2021

Yeast calmodulin (CaM) was found to be localized to the microtubule organizing centre, the spindle pole body. The spindle pole body is a multi-layered structure consisting of outer, central and inner plaques. In this paper, we report that a fraction of CaM is in association with the central plaque of the spindle pole body. This localization is dependent on the calmodulin-binding site of another spindle pole body component, Spc110p, which serves as a spacer connecting the inner plaque with the central plaque. Since the CaM-binding site of Spc110p is located near the carboxy terminus, Spc110p-dependent localization of calmodulin defines the orientation of Spc110p with the carboxy terminus towards the central plaque and the amino terminus towards the inner plaque. This orientation of Spc110p was confirmed using antibodies specific for the amino-terminal end of Spc110p, which predominantly labelled the inner plaque. In addition, synthetic peptides corresponding to the calmodulin-binding site of Spc110p bound to calmodulin with a Kd in the nanomolar range and nearly independent of Ca2+.
Location of the HIV-1 Rev protein during mitosis: Inactivation of the nuclear export signal alters the pathway for postmitotic reentry into nucleoli
Authors: Dundr Miroslav , Leno Gregory H. , Lewis Nancy , Rekosh David , Hammarskjöld Marie-Louise , Olson Mark O. J.
Published: April 2021

The HIV-1 Rev protein localizes predominantly to the nucleolus of HIV-1-infected or Rev-expressing cells. The subcellular location of Rev during mitotic nucleolar disintegration was examined at various stages of mitosis in synchronized Rev-expressing CMT3 cells. During early prophase Rev was predominantly located in disintegrating nucleoli and began to accumulate at the peripheral regions of chromosomes in late prophase, eventually distributing uniformly on all chromosomes in prometaphase. In anaphase Rev remained associated with the perichromosomal regions, but significant amounts of Rev were also seen in numerous nucleolus-derived foci. The movement of Rev from disintegrating nucleoli to perichromosomal regions and foci was similar to that of nonribosomal nucleolar proteins, including fibrillarin, nucleolin, protein B23 and p52 of the granular component. During telophase Rev remained associated with perichromosomal regions and mitotic foci until the nuclear envelope started to reform.

When nuclear envelope formation was complete in late telophase, nonribosomal nucleolar proteins were present in prenucleolar bodies (PNBs) which were eventually incorporated into nucleoli; at the same time, Rev was excluded from nuclei. In contrast, a trans-dominant negative Rev protein containing an inactive nuclear export signal reentered nuclei by the nonribosomal nucleolar protein pathway in late telophase, associating with PNBs and reformed nucleoli. Rev protein reentry into postmitotic nuclei was delayed until early G1 phase, but before the arrival of ribosomal protein S6. Thus, Rev behaves like a nonribosomal nucleolar protein through mitosis until early telophase; however, its nuclear reentry seems to require reestablishment of both a nuclear import system and active nucleoli.
Molecular characterization of mitofilin (HMP), a mitochondria-associated protein with predicted coiled coil and intermembrane space targeting domains
Authors: Odgren Paul R. , Toukatly Gary , Bangs Peter L. , Gilmore Reid , Fey Edward G.
Published: April 2021

We have identified and characterized a human protein of the mitochondria which we call mitofilin. Using monoclonal and polyclonal antibodies, we have isolated cDNA clones and characterized mitofilin biochemically. It appears as a 90 and 91 kDa doublet in western blots and is translated from a single 2.7 kb mRNA. Antibodies raised against cellular and bacterially-expressed protein give identical cytoplasmic immunofluorescence and immunoblot results. Mitofilin co-localizes with mitochondria in immunofluorescence experiments and co-purifies with mitochondria. Double label studies show co-localization only with mitochondria and not with Golgi or endoplasmic reticulum. Colocalization with mitochondria is retained when actin or tubulin are de-polymerized, and mitofilin is expressed in all human cell types tested. The cDNA encodes a polypeptide with a central α-helical region with predicted coiled coil domains flanked by globular amino and carboxy termini. Unlike coiled coil motor proteins, mitofilin is resistant to detergent extraction. The presence of mitochondrial targeting and stop-transfer sequences, along with the accessibility of mitofilin to limited proteolysis suggests that it resides predominantly in the intermembrane space, consistent with immuno-electron micrographs which show mitofilin mainly at the mitochondrial periphery. The cDNA sequence of mitofilin is identical to that recently reported by Icho et al. (1994; Gene 144, 301-306) for a mRNA preferentially expressed in heart muscle (HMP), consistent with the high levels of mitochondria in cardiac myocytes.
Expression, localization and functional role of small GTPases of the Rab3 family in insulin-secreting cells
Authors: Regazzi Romano , Ravazzola Mariella , Iezzi Mariella , Lang Jochen , Zahraoui Ahmed , Andereggen Elisabeth , Morel Philippe , Takai Yoshimi , Wollheim Claes B.
Published: April 2021

We examined the presence of small molecular mass GTP-binding proteins of the Rab3 family in different insulinsecreting cells. Rab3B and Rab3C were identified by western blotting in rat and in human pancreatic islets, in two rat insulin-secreting cell lines, RINm5F and INS-1, as well as in the hamster cell line HIT-T15. In contrast, Rab3A was detected in rat pancreatic islets as well as in the two insulin-secreting rat cell lines but not in human pancreatic islets and was only barely discernible in HIT-T15 cells. These findings were confirmed by two-dimensional gel electrophoresis followed by GTP-overlay of homogenates of pancreatic islets and of the purified protein. Northern blotting analysis revealed that Rab3D is expressed in the same insulin-secreting cells as Rab3A. Separation of the cells of the rat islets by fluorescence-activated cell sorting demonstrated that Rab3A was exclusively expressed in β-cells. Rab3A was found to be associated with insulin-containing secretory granules both by immunoflu-orescence, immunoelectron microscopy and after sucrose density gradient. Overexpression in HIT-T15 cells of a Rab3A mutant deficient in GTP hydrolysis inhibited insulin secretion stimulated by a mixture of nutrients and bombesin. Insulin release triggered by these secretagogues was also slightly decresed by the overexpression of wildtype Rab3A but not by the overexpression of wild-type Rab5A and of a Rab5A mutant deficient in GTP hydrolysis. Finally, we studied the expression in insulin-secreting cells of rabphilin-3A, a putative effector protein that associates with the GTP-bound form of Rab3A. This Rab3A effector was not detectable in any of the cells investigated in the present study. Taken together these results indicate an involvement of Rab3A in the control of insulin release in rat and hamster. In human β-cells, a different Rab3 isoform but with homologous function may replace Rab3A.
Nuclear lamina and nuclear matrix organization in sperm pronuclei assembled in Xenopus egg extract
Authors: Zhang Chuanmao , Jenkins Hazel , Goldberg Martin W. , Allen Terry D. , Hutchison Christopher J.
Published: April 2021

Nuclear lamina and matrices were prepared from sperm pronuclei assembled in Xenopus egg extracts using a fractionation and extraction procedure. Indirect immunofluorescence revealed that while chromatin was efficiently removed from nuclei during the extraction procedure, the distribution of lamins was unaffected. Consistent with this data, the ammount of lamin B3, determined by immunoblotting, was not affected through the extraction procedure. Nuclear matrices were visualised in DGD sections by TEM. Within these sections filaments were observed both at the boundary of the nucleus (the lamina) and within the body of the nucleus (internal nuclear matrix filaments). To improve resolution, nuclear matrices were also prepared as whole mounts and viewed using field emission in lens scanning electron microscopy (FEISEM). This technique revealed two distinct networks of filaments. Filaments lying at the surface of nuclear matrices interconnected nuclear pores. These filaments were readily labelled with monoclonal anti-lamin B3 antibodies. Filaments lying within the body of the nuclear matrix were highly branched but were not readily labelled with antilamin B3 antibodies. Nuclear matrices were also prepared from sperm pronuclei assembled in lamin B3 depleted extracts. Using FEISEM, filaments were also detected in these preparations. However, these filaments were poorly organised and often appeared to aggregate. To confirm these results nuclear matrices were also observed as whole mounts using TEM. Nuclear matrices prepared from control nuclei contained a dense array of interconnected filaments. Many (but not all) of these filaments were labelled with anti-lamin B3 antibodies. In contrast, nuclear matrices prepared from ‘lamin depleted nuclei’ contained poorly organised or aggregated filaments which were not specifically labelled with anti-lamin B3 antibodies.
Putative GTPase Gtr1p genetically interacts with the RanGTPase cycle in Saccharomyces cerevisiae
Authors: Nakashima Nobutaka , Hayashi Naoyuki , Noguchi Eishi , Nishimoto Takeharu
Published: April 2021

In order to identify a protein interacting with RCC1, a guanine nucleotide-exchange factor for the nuclear GTPase Ran, we isolated a series of cold-sensitive suppressors of mtr1-2, a temperature-sensitive mutant of the Saccharomyces cerevisiae RCC1 homologue. One of the isolated suppressor mutants was mutated in the putative GTPase Gtr1p, being designated as gtr1-11. It also suppressed other alleles of mtr1-2, srm1-1 and prp20-1 in contrast to overexpression of the S. cerevisiae Ran/TC4 homologue Gsp1p, previously reported to suppress prp20-1, but not mtr1-2 or srm1-1. Furthermore, gtr1-11 suppressed the rna1-1, temperature-sensitive mutant of the Gsp1p GTPase-activating protein, but not the srp1-31, temperature-sensitive mutant of the S. cerevisiae importin α homologue. mtr1-2, srm1-1 and prp20-1 were also suppressed by overexpression of the mutated Gtr1p, Gtr1-11p. In summary, Gtr1p that was localized in the cytoplasm by immunofluoresence staining was suggested to function as a negative regulator for the Ran/TC4 GTPase cycle.
Engagement of L-selectin impairs the actin polymerizing capacity of β2 integrins on neutrophils
Authors: Ng-Sikorski Janet , Lindén Lotta , Eierman David , Franzen Lennart , Molony Leslie , Andersson Tommy
Published: April 2021

A sequential activation of L-selectin and β2-integrins on neutrophils is crucial for the rolling, adherence and subsequent migration of these cells on the endothelium. However, little is known about a possible interplay between these adhesion receptors in the final regulation of cell motility. The results presented here show that sulfatides themselves (here used as tools to activate L-selectins), have no major effect on the cellular content of filamentous actin (F-actin), but cause a time-related decrease in the β2-integrin-induced formation of F-actin. This effect of sulfatides was abolished in cells lacking L-selectin as a result of pretreatment with chymotrypsin. A similar sulfatide-induced activation of Lselectin also caused a pronounced and time-related decrease of a subsequent chemotactic peptide-induced F-actin response. The effect of sulfatides on both β2-integrin- and chemotactic peptide-induced F-actin were abolished if Lselectin were blocked by preincubating the cells with specific antibodies to L-selectin. These effects of L-selectin engagement on cellular F-actin content were neither abolished by blocking the cytosolic free Ca2+ signal with bis(2-amino-5-methylphenoxy)ethane-N,N,N’,N’-tetraacetic-acid tetraacetoxymethyly ester (MAPT/AM) nor by blocking a cAMP-induced activation of protein kinase A by pretreating the cells with adenosine-3’,5’-cyclic monophosphorothioate (Rp-cAMPS). Instead we found that L-selectin engagement impaired an early β2-integrin-induced tyrosine kinase activation, an event shown to be necessary for a normal β2-integrin-mediated F-actin response. The present demonstration of a negative feed-back function of L-selectin on β2-integrin-induced modulations of the actin cytoskeleton, suggests that the relative distribution and/or density of the respective L-selectin and β2-integrin ligands on endothelial cells might be important factors in determining the final site of firm adhesion and extravasation of neutrophils.
Mitosis in heterobasidiomycetous yeasts: II. Rhodosporidium sp. (Rhodotorula glutinis) and Aessosporon salmonicolor (Sporobolomyces salmonicolor)
Authors: Mccully E. Kathleen , Robinow C. F.
Published: April 2021

Mitotic events similar to those in Leucosporidium scottii also take place in 2 other hetero-basidiomycetous yeasts – Rhodosporidium sp. and Aessosporon salmonicolor, which were formerly known as Rhodotorula glutinis and Sporobolomyces salmonicolor. Therefore it is proposed that a mitosis which takes place within the bud may be a distinctive characteristic of heterobasidio-mycetous yeasts which can be used as a taxonomic indicator of basidiomycete relationships in asporogenous yeasts.

Mitosis in A. salmonicolor seems to be identical to that which we have previously described in L. scottii but a few differences have been found in Rhodosporidium sp. These are now described in detail.
Structures of physiological interest in the frog heart ventricle
Authors: Page Sally G. , Niedergerke R.
Published: April 2021

Two structures of physiological interest in frog heart ventricles have been examined in detail: (a) the layer of endothelial cells which encloses each bundle of heart fibres, and (b) the sarcoplasmic reticulum (SR) inside the heart fibres. Some additional observations on fibre sizes and types have been made.

Movement across the endothelial cell layer of molecules (molecular or ionic size ≤ 12·5 nm) occurs through narrow clefts separating each endothelial cell from its neighbour. This conclusion results from experiments made with the extracellular markers ferritin and horseradish peroxidase.

A diffusion equation describing the movement of solutes into and out of the fibre bundle has been derived using several geometrical parameters, such as the length and width of the clefts and the size of the extracellular aqueous space inside the bundle, all of which were determined from electron micrographs of the tissue.

The theoretical solution for a stepwise change of external calcium concentration gives a half-time of 2·3 s (± 0·8 s, s.D. of 13 bundles) for diffusion equilibrium at the surface of the heart fibres; this value, however, is likely to be an overestimate, by some 20–30%, on account of several systematic errors which are described.

The sarcoplasmic reticulum in heart fibres consists of a network of thin tubules which partially encircle the myofibrils at Z-line level and also form occasional longitudinal connexions. Branches extend to peripheral regions of the cell and terminate in close apposition to the inner surface of the cell membrane. The volume of the SR is estimated to be approximately 0-5 % of the myofibrillar volume of the cells.

Cross-sectional areas of heart fibres (and also their shapes) vary considerably, from less than 2 to more than 100 μm2 (average 17·4 μm2). Fibres of large size and small surface/volume ratio contain many fewer myofibrils and more glycogen granules than fibres of the same size but larger surface/volume ratio.

Physiological implications of these results are discussed.
On the physical identity of scintillons: Bioluminescent particles in Gonyaulax polyedra
Authors: Fogel Margaret , Schmitter Ruth E. , Hastings J. W.
Published: April 2021

The physical identity of the bioluminescent particles (‘scintillons’) extracted from the marine dinoflagellate Gonyaulax polyedra was investigated by electron microscopy and particle counts of the active fractions from sucrose density gradients. Membranous structures are abundantly present in purified fractions, while there is a decrease, relative to activity, in the numbers of guanine crystals, which had previously been implicated as bioluminescent structures. The significance of these findings with respect to the in vivo scinrillon is discussed.
Origin and structure of embryoids arising from epidermal cells of the stem of Ranunculus sceleratus l
Authors: Konar R. N. , Thomas E. , Street H. E.
Published: April 2021

Embryoids are initiated from single highly cytoplasmic cells of the stem epidermis of plantlets of Ranunculus sceleratus L. derived from callus cultures. The embryoids show embryological development of the Crucifer type except that the suspensor is reduced or suppressed. The cells of the young embryoids have a large central nucleus surrounded by small vacuoles, a cytoplasm rich in free ribosomes or ribosomes in small polysomes, prominent amyloplastids and abundant spherosomes. These cells also frequently contain multivesicular and myelin bodies. The spherosomes are observed in various stages of development; they are typically arranged in a layer below the cell wall, the space between the spherosomes and the wall being rich in spherical vesicles and microtubules. As embryoid development proceeds the plastids show internal differentiation; globular centres and, surrounding them, electron-transparent droplets are associated with the origin of the thylakoid membranes. The walls of the embryogenic cells are initially rich in plasmodesmata but as the embryoid develops cytoplasmic continuity with the embedding tissue is severed and the embryoid is clearly delineated. The embryology and fine structure of the embryoid cells is compared with that of zygotic embryos of Capsella and Ranunculus.
Induced root differentiation in sycamore callus
Authors: Wright K. , Northcote D. H.
Published: April 2021

A sycamore callus (S 4) has been isolated and grown on a medium containing salts, vitamins, a casein digest, 2 % sucrose and 1 mg/1. NAA. The callus, which would not grow in the absence of the added auxin, was much firmer in texture than a sycamore callus (S 2) isolated in this laboratory in 1958 which has not been induced to differentiate. When kinetin over the range 0·05–0·5 mg/1. was included in the growth medium of S 4 nodules of xylem and phloem were induced within the tissue and roots frequently grew from the surface of the callus. Some roots developed geotropic sensitivity although the majority grew radially outwards from the callus surface. The roots also varied with respect to the number of root hairs they carried. No roots were produced at sucrose concentrations less than 2%, although histological examination revealed extensive xylem and phloem differentiation relative to the amount of growth which had taken place. When sugars other than sucrose were supplied in the medium at a concentration of 3 % (w/v) roots were also induced in those calluses where the carbon source had supported good growth. Sucrose, glucose and fructose were identified in the ethanol-soluble extracts of all these calluses. Radioactivity was incorporated into sucrose when S 4 was incubated on a medium containing D-[U-14C]glucose for 24 h.

Any sugar which supported growth and differentiation was therefore one which was capable of entering the common metabolic pathway used by the plant for glucose and sucrose. The cells could undergo differentiation so long as the sugar they were supplied with supported active growth and division. The possibility of a physiological role for sucrose is discussed.
The basis of cell-to-cell transformation in Paramecium bursaria: I. transfer of cytoplasmic material
Authors: Cullis C. A.
Published: April 2021

A mating type transformation, involving cell-to-cell contact (abortive conjugation) in syngen 4 of Parameciwn bursaria has been investigated. By autoradiography, transfer of material between the conjugants in normal and abortive conjugation has been shown to occur. The transfer of this material, which includes protein, RNA and DNA, during abortive conjugation has been shown to be necessary if the transformation is to occur.

The mating type substances produced by sexually competent cells of different mating types have been characterized with respect to their thermal stability. In all cases examined there appeared to be 3 mating type substances produced which had different temperature stabilities. No differences could be shown between the transformed cells and normal cells of the same mating type in relation to stability of mating type substance.
The Organization and Polarity of Pollen Mother Cells of Triticum Aestivum
Authors: Dover G. A.
Published: April 2021

Colchicine has been applied to young developing anthers of Triticum aestivuni at varying stages of maturity from the last premeiotic mitoses of the archesporial and tapetai cells to the second meiotic divisions of the pollen mother cells. The developmental stage of the archesporium at which colchicine took effect was determined by cytological examination of the ploidy levels of the nuclei of the adjacent tapetai cells. The type of pollen abnormality induced depended on the time of application and the concentration of colchicine. Uninucleate monads with 4 randomly positioned pores and uninucleate monads without pores were obtained with 0·5 % colchicine. Multipored polyads and multipored uninucleate monads were observed together in anthers treated with 0·01 % colchicine. Naturally occurring aberrant pollen types in hybrids of Triticum aestivuni × Aegilops rnutica or T. aestivum x Aegilops sharonensis have revealed a constant relationship between the disposition of the meiotic spindles and the siting of the pollen pores. The colchicine-induced abnormalities have further clarified the nature of this relationship leading to the interpretation that both the positioning of the spindles and the siting of the pores are predetermined by events taking place in the premeiotic interphase at a time just after the last mitosis of the pollen mother cells and the penultimate mitosis of the tapetum. A reorganization of the archesporial cells (sensitive to colchicine) possibly occurs at this time. Various subsequent meiotic events are dependent on the reorganization. Two of these events - the organization of meiotic spindles and the establishment of pollen symmetry - are discussed.
Timing of Nucleolar Activity in Meristems
Authors: Torre C. De La , Clowes F. A. L.
Published: April 2021

The nucleoli in 4 regions of the root meristem of Zea mays have been examined and the time spent in each of the phases of the nucleolar cycle has been calculated from estimates of the rates of mitosis and the fraction of cycling cells in each region. The nucleolar cycles have been fitted to the mitotic cycles of cycling cells in the same regions.

The timing of activity of the r-RNA genes differs in relation to the chromosomal cycles of condensation and movement in the different regions of the meristem. In the stelar regions disorganization of the nucleolus starts in the middle of prophase and reorganization starts towards the end of telophase and is completed in the following interphase. In the quiescent centre of the meristem the phases of both cycles are lengthened except for telophase and reorganization of the nucleolus does not start until after the end of telophase. The acceleration of telophase in the quiescent centre is discussed in relation to inhibition of protein synthesis. In the adjacent cap initials, which have the shortest mitotic cycle, the nucleolus starts to disorganize before the onset of prophase and reorganization is completed halfway through telophase. This fits with the fact that DNA synthesis is advanced into the telophase of the previous cycle in these cells.
Symmetric and Asymmetric Mitosis and Cytokinesis in the Root Tip of Hydrocharis Morsus-Ranae L.
Authors: Cutter Elizabeth G. , Hung Ching-Yuan
Published: April 2021

In the roots of Hydrocharis morsus-ranae, certain cells of the protoderm divide asymmetrically to form a small, highly cytoplasmic trichoblast proximally, and a larger, more vacuolate epidermal cell distally. The former develops as a root hair without further division; the latter divides several times to form ordinary epidermal cells. During mitosis, presumed dictyosome vesicles and fragments or sections of reticulated or serrate sheets of ER, aligned with the spindle microtubules, were observed among the chromosomes as early as metaphase, suggesting that the portions of ER were involved in formation of the cell plate or in some other function in the equatorial region. A pre-prophase band of microtubules was not observed. Asymmetric divisions differ from symmetric ones in the skewed orientation of the metaphase plate, the formation of a curved, rather wavy cell wall and the slightly greater vacuolation of one daughter cell. Less difference in the ultrastructure of the daughter cells resulting from an asymmetric division was observed in this rather slowly growing material than in other examples previously described in the literature.
A Gradient of Positional Information in an Insect, Rhodnius
Authors: Lawrence P. A. , Crick F. H. C. , Munro M.
Published: April 2021

Locke discovered a segmental gradient in Rhodnius which controls the polarity of the epidermal cell and gives positional information. The polarity is expressed by the orientation of folds in the adult epicuticle, which are aligned parallel to the contours in the gradient. It was later suggested that this gradient could be of a concentration of a diffusible substance. Because concentration gradients could be maintained in various ways we have simulated several models in the computer, and examined the results of rotating square pieces of model landscape through 90° and allowing diffusion. The gradient landscapes after different times and at equilibrium are plotted as contour maps and are compared with cuticle patterns from adult insects after rotation of square pieces of epidermis in larvae.

One simple model, where the gradient depends only on the activities of a line of source cells at one end of the segment and a line of sink cells at the other, is eliminated by 2 observations: (1) the theoretical and experimental patterns are consistently different; and (2) when adults developing from operated larvae are made to form a supernumerary cuticle the first and second cuticles have almost identical patterns. This suggests that the gradient landscape has reached a steady state.

In another model the cells are considered to act as homeostatic units in the gradient, and when moved to a new position they each attempt to maintain their original or ‘set’ concentration. Simulation of this model gives equilibrium patterns which are similar to the experimental results. It is suggested that the cells become ‘set’ at some stage in the cell cycle to the ambient concentration. This hypothesis predicts that after reaching initial equilibrium the pattern should change only if there are cell divisions. Adult insects are made to moult again under different conditions and it is found that pattern change is correlated with cell divisions.

Locke also observed an asymmetry in the patterns after rotation of squares through 180°. Simulation showed that such asymmetry would result from each cell acting as a better homeo-static unit when moved one way in the gradient (for example when acting as a sink) than when moved the other (acting as a source).

We do not claim that these comparisons eliminate all other classes of model, and present our conclusions in as general a form as possible.
Patterns of Particle Movement in Nerve Fibres In Vitro-An Analysis by Photokymography and Microscopy
Authors: Berlinrood M. , McGee-Russell S. M. , Allen R. D.
Published: April 2021

Tissue-cultured nerve fibres derived from 3 species of amphibian embryos were composed of bundles of neurites, when viewed by electron microscopy, and contained particles moving in both directions by saltations. Photokymographic analysis of ciné microscopy records taken with Zeiss differential interference optics demonstrated instantaneous particle velocities ranging from 0·15 to 1·8 μm s-1 (13–155 mm/day) in Rana pipiens cultures; 0·16–0·8 μm s-1 (14–74 mm/day) in Ambystoma tigrinum cultures and 0·6—2·25 μm s-1 (45—194 mm/day) in Xenopus laevis cultures. Measured pathlengths for particles saltations ranged from 4 to 63 μm. No correlations between particle velocity and pathlength were found.
Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching
Authors: Glogauer M. , Arora P. , Yao G. , Sokholov I. , Ferrier J. , McCulloch C. A. G.
Published: April 2021

The actindependent sensory and response elements of stromal cells that are involved in mechanical signal transduction are poorly understood. To study mechanotransduction we have described previously a collagenmagnetic bead model in which application of well-defined forces to integrins induces an immediate (<1 second) calcium influx. In this report we used the model to determine the role of calcium ions and tyrosine-phosphorylation in the regulation of force-mediated actin assembly and the resulting change in membrane rigidity. Collagen-beads were bound to cells through the focal adhesion-associated proteins talin, vinculin, α2-integrin and β-actin, indicating that force application was mediated through cytoskeletal elements. When force (2 N/m2) was applied to collagen beads, confocal microscopy showed a marked vertical extension of the cell which was counteracted by an actin-mediated retraction. Immunoblotting showed that force application induced F-actin accumulation at the bead-membrane complex but vinculin, talin and α2-integrin remained unchanged. Atomic force microscopy showed that membrane rigidity increased 6-fold in the vicinity of beads which had been exposed to force. Force also induced tyrosine phosphorylation of several cytoplasmic proteins including paxillin. The force-induced actin accumulation was blocked in cells loaded with BAPTA/AM or in cells preincubated with genistein, an inhibitor of tyrosine phosphorylation. Repeated force application progressively inhibited the amplitude of force-induced calcium ion flux. As force-induced actin reorganization was dependent on calcium and tyrosine phosphorylation, and as progressive increases of filamentous actin in the submembrane cortex were correlated with increased membrane rigidity and dampened calcium influx, we suggest that cortical actin regulates stretch-activated cation permeable channel activity and provides a desensitization mechanism for cells exposed to repeated long-term mechanical stimuli. The actin response may be cytoprotective since it counteracts the initial force-mediated membrane extension and potentially strengthens cytoskeletal integrity at force-transfer points.