bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal and spatial aspects of the gradual migration of primordial germ cells from the epiblast into the germinal crescent in the avian embryo
Authors: Ginsburg Malka , Eyal-Giladi Hefzibah
Published: April 2021

The migration of the PGCs from a stage XIII epiblast into the germinal crescent of a stage 10 chick blastoderm was experimentally investigated. Considerable numbers of PGCs start to come down from the epiblastic layer at stage XII–XIII and continue to do so in relatively smaller numbers until stage 6. The earliest PGCs land on the primary hypoblast and probably are carried by it into the GC. The PGCs that descend at relatively later stages land on the mesodermal wings and are either carried by them passively, or move actively into the GC. After the removal of the lower layer from stage 4 and older blastoderms, there is no regeneration of a GC in the blastoderm. Other consequences of this operation are that the mesodermal wings do not spread anteriorly as in normal controls and the PGCs that are still in the mesodermal domain are trapped. PGCs were found in expiants of the lower layer and of the mesoderm. No identifiable PGCs were encountered in expiants of the PS.
Formation of pattern in regenerating tissue pieces of Hydra attenuata
Authors: Bode Patricia Macauley , Bode Hans R.
Published: April 2021

The tentacles in hydra have characteristics of both spacing patterns and number-regulating patterns in that their number under some circumstances changes with the size of the animal and under others does not. To determine which type of processes could yield these results, an extensive analysis was undertaken of the size parameters pertinent to tentacle formation. To do this pieces of tissue, varying in shape and spanning a 30-fold size range, were excised and allowed to regenerate into complete animals. Three separate mechanisms were found to combine which resulted in the final number of tentacles: (1) the spread of the two-part head pattern to produce a competent band of tissue of a given size where the tentacles could form; (2) initiation of tentacle-forming centres by a spacing mechanism and (3) growth of the tentacles to a size proportional to the size of the animal.
Interactions between somite cells: the formation and maintenance of segment boundaries in the chick embryo
Authors: Stern Claudio D. , Keynes Roger J.
Published: April 2021

We have investigated the interactions between the cells of the rostral and caudal halves of the chick somite by carrying out grafting experiments. The rostral halfsclerotome was identified by its ability to support axon outgrowth and neural crest cell migration, and the caudal half by the binding of peanut agglutinin and the absence of motor axons and neural crest cells. Using the chick-quail chimaera technique we also studied the fate of each half-somite.

It was found that when half-somites are placed adjacent to one another, their interactions obey a precise rule: sclerotome cells from like halves mix with each other, while those from unlike halves do not; when cells from unlike halves are adjacent to one another, a border is formed.

Grafting quail half-somites into chicks showed that the fates of the rostral and caudal sclerotome halves are similar: both give rise to bone and cartilage of the vertebral column, as well as to intervertebral connective tissue. We suggest that the rostrocaudal subdivision serves to maintain the segmental arrangement when the mesenchymal sclerotome dissociates, so that the nervous system, vasculature and possibly vertebrae are patterned correctly.
Patterns of dna replication in Drosophila polytene nuclei replicating in Xenopus egg and oocyte extracts
Authors: Sleeman Andrea M. , Leno Gregory H. , Mills Anthony D. , Fairman Micaela P. , Laskey Ronald A.
Published: April 2021

We have used Xenopus laevis cell-free extracts to study patterns of DNA replication in polytene nuclei isolated from salivary glands of Drosophila melanogaster 3rd instar larvae. Replication was visualized by supplementation with biotin-dUTP so that nascent DNA became labelled, thus allowing detection with fluorescein or Texas-Red-conjugated streptavidin.

Biotin incorporation was dependent on incubation in extracts. Transverse bands were labelled in high-speed supernatants of eggs or oocytes in which replication does not initiate de novo. These patterns corresponded to the patterns of endogenous replication forks in polytene nuclei, monitored by bromodeoxyuridine incorporation in intact salivary glands. By contrast, when nuclei were incubated in low-speed supernatants of eggs, they underwent more extensive chromatin decondensation and initiated replication. The spatial patterns of replication are strikingly different from the endogenous patterns. Instead they closely resemble patterns of clustered replication forks seen in Xenopus sperm nuclei replicating in the extract. This indicates that the egg extract can impose its pattern of replication foci even when the template is presented in the highly organized form of a polytene nucleus.
Redistribution of nuclear lamin A is an early event associated with differentiation of human promyelocytic leukemia HL-60 cells
Authors: Collard Jean-François , Senécal Jean-Luc , Raymond Yves
Published: April 2021

The nuclear lamina of mammalian somatic cells is characterized by the constitutive presence of lamin B polypeptides while the appearance of lamins A and C generally occur during establishment of a differentiated phenotype. We have used antibodies specific to the unique carboxy-terminal domain of lamin A, i.e. distinct from the shared domains of lamins A and C, to study the individual behaviour of lamin A during establishment of a macrophage phenotype in human HL-60 cells. Lamin A was present as a nuclear cap in the majority of undifferentiated cells and it was redistributed to a full peripheral nuclear location very early after induction of differentiation by phorbol esters, even in the presence of a protein synthesis inhibitor. Induction of the cells into a reversible precommitment state by bromodeoxyuridine was accompanied by a similar redistribution of lamin A that however reverted to a cap after removal of inducer. No changes were observed in the uniform peripheral nuclear location of lamin C under all of these conditions. These results strongly suggest that lamin A plays a role in the early events of cell differentiation. Taken together with previous results on the interaction of A-type lamins with chromatin, these findings offer experimental evidence consistent with the proposed role of A-type lamins, and particularly lamin A, in the process of chromatin reorganization that accompanies the expression of a differentiated phenotype.
Differential gene expression during capillary morphogenesis in 3D collagen matrices
Authors: Bell Scott E. , Mavila Anil , Salazar René , Bayless Kayla J. , Kanagala Suhasini , Maxwell Steven A. , Davis George E.
Published: April 2021

We have performed a screening analysis of differential gene expression using a defined in vitro model of human capillary tube formation. Gene array, differential display and cDNA library screening were used to identify both known and novel differentially expressed genes. Major findings include: the upregulation and functional importance of genes associated with basement membrane matrix assembly; the upregulation of growth factors, transcription factors, anti-apoptotic factors, markers of endothelial cell differentiation, JAK-STAT signalling molecules, adhesion receptors, proteinase inhibitors and actin regulatory proteins; and expression changes consistent with inhibition of cell cycle progression, increased cholesterol biosynthesis, decreased ubiquitin-proteasome mediated degradation, and activation of G-protein signaling pathways. Using DNA microarray analysis, the most induced genes at 8, 24 and 48 hours compared with those at 0 hours were jagged-1, stanniocalcin and angiopoietin-2, whereas the most repressed genes were connective tissue growth factor, fibulin-3 and RGS-5. In addition, the full length coding sequence of two novel regulated capillary morphogenesis genes (CMGs) are presented. CMG-1 encodes a predicted intracellular 65 kDa protein with coiled-coil domains. A CMG-1-green fluorescent protein (GFP) chimera was observed to target to an intracellular vesicular compartment. A second novel gene, CMG-2, was found to encode a predicted intracellular protein of 45 kDa containing a transmembrane segment and a CMG-2-GFP chimera was observed to target to the endoplasmic reticulum. A recombinant portion of CMG-2 was found to bind collagen type IV and laminin, suggesting a potential role in basement membrane matrix synthesis and assembly. These data further elucidate the genetic events regulating capillary tube formation in a 3D matrix environment.
Arylhydrocarbon receptor (AhR) is involved in negative regulation of adipose differentiation in 3T3-L1 cells
Authors: Shimba Shigeki , Wada Taira , Tezuka Masakatsu
Published: April 2021

The arylhydrocarbon receptor (AhR) is the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although a physiological ligand for the AhR has yet to be identified, and the precise physiological roles of the AhR are unknown, it may play important roles not only in the regulation of xenobiotic metabolism but also in the maintenance of homeostatic functions. We have previously reported that the level of AhR protein decreased with ongoing adipose differentiation in 3T3-L1 cells. Studies using a TCDD-resistant clone of 3T3-L1 cells suggested that the AhR may be involved in the negative regulation of adipose differentiation. To confirm this hypothesis, 3T3-L1 fibroblast cells were stably transfected with a vector expressing high levels of full-length sense AhR mRNA, antisense AhR mRNA or a control vector. Comparison of the differentiation potency of these clones with that of control cells showed that overexpression of the AhR suppressed morphological differentiation, as well as induction of adipocyte-related genes, whereas decreased expression of the AhR induced much greater morphological differentiation and expression of adipocyte-related genes. Activation of PPARγ2 with ligands such as troglitazone, ciglitazone and indomethacin restored the ability of the AhR-overexpressing cells to differentiate. The cells overexpressing the AhR exhibited the higher p42/p44 MAP kinase activity compared with the control cells. Treatment with PD98059 or U0126 also abrogated the inhibitory action of the AhR on adipogenesis. We also present data showing that activation of the AhR slowed clonal expansion. During clonal expansion, the AhR inhibited the pRB phosphorylation and the downregulation of p107 expression. Taken together, these results strongly suggest that AhR is a negative regulator of adipose differentiation in 3T3 L1 cells.
Alcohol oxidase and dihydroxyacetone synthase, the abundant peroxisomal proteins of methylotrophic yeasts, assemble in different cellular compartments
Authors: Stewart Mary Q. , Esposito Renee D. , Gowani Jehangir , Goodman Joel M.
Published: April 2021

Alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS) constitute the bulk of matrix proteins in methylotrophic yeasts, model organisms for the study of peroxisomal assembly. Both are homooligomers; AO is a flavin-containing octamer, whereas DHAS is a thiamine pyrophosphate-containing dimer. Experiments in recent years have demonstrated that assembly of peroxisomal oligomers can occur before import; indeed the absence of chaperones within the peroxisomal matrix calls into question the ability of this compartment to assemble proteins at all. We have taken a direct pulse-chase approach to monitor import and assembly of the two major proteins of peroxisomes in Candida boidinii. Oligomers of AO are not observed in the cytosol, consistent with the proteins inability to undergo piggyback import. Indeed, oligomerization of AO can be followed within the peroxisomal matrix, directly demonstrating the capacity of this compartment for protein assembly. By contrast, DHAS quickly dimerizes in the cytosol before import. Binding and import was slowed at 15°C; the effect on AO was more dramatic. In conclusion, our data indicate that peroxisomes assemble AO in the matrix, while DHAS undergoes dimerization prior to import.
Post-transcriptional gene silencing in plants
Authors: Vaucheret Hervé , Béclin Christophe , Fagard Mathilde
Published: April 2021

Post-transcriptional gene silencing (PTGS) in plants is an RNA-degradation mechanism that shows similarities to RNA interference (RNAi) in animals. Indeed, both involve double-stranded RNA (dsRNA), spread within the organism from a localised initiating area, correlate with the accumulation of small interfering RNA (siRNA) and require putative RNA-dependent RNA polymerases, RNA helicases and proteins of unknown functions containing PAZ and Piwi domains. However, some differences are evident. First, PTGS in plants requires at least two genes – SGS3 (which encodes a protein of unknown function containing a coil-coiled domain) and MET1 (which encodes a DNA-methyltransferase) – that are absent in C. elegans and thus are not required for RNAi. Second, all Arabidopsis mutants that exhibit impaired PTGS are hypersusceptible to infection by the cucumovirus CMV, indicating that PTGS participates in a mechanism for plant resistance to viruses. Interestingly, many viruses have developed strategies to counteract PTGS and successfully infect plants – for example, by potentiating endogenous suppressors of PTGS. Whether viruses can counteract RNAi in animals and whether endogenous suppressors of RNAi exist in animals is still unknown.
Adenovirus-Cre-mediated recombination in mammary epithelial early progenitor cells
Authors: Rijnkels Monique , Rosen Jeffrey M.
Published: April 2021

The transplantation of primary mammary epithelial cells after adenovirus-Cre-mediated recombination provides a new approach for the study of specific gene function during mammary gland development and in breast cancer. Most mammary-gland-specific promoters identified to date are regulated by lactogenic hormones. They are expressed predominantly in lobuloalveolar cells during pregnancy and lactation, but not during early stages of ductal morphogenesis in the mammary epithelial cell progenitors, which are primarily implicated in tumorigenesis. In transgenic mice these promoters will continually or repeatedly express Cre depending on the hormonal environment precluding the definition of cell lineages. To circumvent these limitations, we have taken advantage of the unique regenerative capacity of mammary epithelium to reconstitute a mammary gland in an epithelium-cleared fat pad in conjunction with transient Cre expression using recombinant adenovirus in primary cultures. This approach was validated using mice carrying reporter constructs that exclusively express the LacZ gene after Cre-mediated deletion of a floxed DNA fragment. These studies demonstrated that, following recombination, cells that are marked as genetically manipulated contribute to the reconstitution of the mammary gland. The presence of β-galactosidase-expressing cells in serial transplants of the primary outgrowths indicated that early progenitor or stem cells were successfully targeted. With the increased availability of floxed alleles, this approach should greatly facilitate the study of gene function during early stages of mammary gland development and in breast cancer.
Molecular complexity and dynamics of cell-matrix adhesions
Authors: Zamir Eli , Geiger Benjamin
Published: April 2021

Currently >50 proteins have been reported to be associated with focal contacts and related ECM adhesions. Most of these contain multiple domains through which they can interact with different molecular partners, potentially forming a dense and heterogeneous protein network at the cytoplasmic faces of the adhesion site. The molecular and structural diversity of this ‘submembrane plaque’ is regulated by a wide variety of mechanisms, including competition between different partner proteins for the same binding sites, interactions triggered or suppressed by tyrosine phosphorylation, and conformational changes in component proteins, which can affect their reactivity. Indeed, integrin-mediated adhesions can undergo dynamic changes in structure and molecular properties from dot-like focal complexes to stress-fiber-associated focal contacts, which can further ‘mature’ to form fibronectin-bound fibrillar adhesions. These changes are driven by mechanical force generated by the actin- and myosin-containing contractile machinery of the cells, or by external forces applied to the cells, and regulated by matrix rigidity.
Isolation and characterization of the Pin1/Ess1p homologue inSchizosaccharomyces pombe
Authors: Huang Han-kuei , Forsburg Susan L. , John Ulrik P. , O’Connell Matthew J. , Hunter Tony
Published: April 2021

Pin1/Ess1p is a highly conserved WW domain-containing peptidyl-prolyl isomerase (PPIase); its WW domain binds specifically to phospho-Ser/Thr-Pro sequences and its catalytic domain isomerizes phospho-Ser/Thr-Pro bonds. Pin1 PPIase activity can alter protein conformation in a phosphorylation-dependent manner and/or promote protein dephosphorylation. Human Pin1 interacts with mitotic phosphoproteins, such as NIMA, Cdc25 and Wee1, and inhibits G2/M progression in Xenopus extracts. Depletion of Pin1 in HeLa cells and deletion of ESS1 in S. cerevisiae result in mitotic arrest. In addition, Pin1/Ess1p play roles in transcription in S. cerevisiae and in mammalian somatic cells. The S. pombe genome sequence has an open reading frame (ORF) that has 47% identity with Pin1. Expression of this ORF rescued the growth defect caused by ess1 deletion in S. cerevisiae, indicating that S. pombe Pin1p is a functional Pin1 homologue. Overexpression of pin1+ in S. pombe caused slow growth and a G1 delay. Deletion of pin1+ (pin1Δ) did not affect cell cycle progression or cell growth, but increased sensitivity to the cyclophilin inhibitor, cyclosporin A, suggesting that cyclophilin family PPIases have overlapping functions with the Pin1p PPIase. Deletion of pin1+ did not affect the DNA replication checkpoint, but conferred a modest increase in UV sensitivity. Furthermore, the pin1Δ allele caused a synthetic growth defect when combined with either cdc25-22 or wee1-50 but not the cdc24-1 temperature-sensitive mutant. The pin1Δ strain showed increased sensitivity to the PP1/PP2A family phosphatase inhibitor, okadaic acid, suggesting that Pin1p plays a role in protein dephosphorylation as a result of its ability to increase the population of phospho-Ser/Thr-Pro peptide bonds in the trans conformation that is required for PP2A-mediated dephosphorylation. Our genetic data also suggest that Pin1p might function as a positive regulator of Cdc25p and Wee1p.
ABC transporters required for endocytosis and endosomal pH regulation inDictyostelium
Authors: Brazill Derrick T. , Meyer Lowell R. , Hatton R. Diane , Brock Debra A. , Gomer Richard H.
Published: April 2021

In Dictyostelium, the RtoA protein links both initial cell-type choice and physiological state to cell-cycle phase. rtoA– cells (containing a disruption of the rtoA gene) generally do not develop past the mound stage, and have an abnormal ratio of prestalk and prespore cells. RtoA is also involved in fusion of endocytic/exocytic vesicles. Cells lacking RtoA, although having a normal endocytosis rate, have a decreased exocytosis rate and endosomes with abnormally low pHs. RtoA levels vary during the cell cycle, causing a cell-cycle-dependent modulation of parameters such as cytosolic pH (Brazill et al., 2000). To uncover other genes involved in the RtoA-mediated differentiation, we identified genetic suppressors of rtoA. One of these suppressors disrupted two genes, mdrA1 and mdrA2, a tandem duplication encoding two members of the ATP binding cassette (ABC) transporter superfamily. Disruption of mdrA1/mdrA2 results in release from the developmental block and suppression of the defect in initial cell type choice caused by loss of the rtoA gene. However, this is not accomplished by re-establishing the link between cell type choice and cell cycle phase. MdrA1 protein is localized to the endosome. mdrA1–/mdrA2– cells (containing a disruption of these genes) have an endocytosis rate roughly 70% that of wild-type or rtoA– cells, whereas mdrA1–/mdrA2–/rtoA– cells have an endocytosis rate roughly 20% that of wild-type. The exocytosis rates of mdrA1–/mdrA2– and mdrA1–/mdrA2–/rtoA– are roughly that of wild-type. mdrA1–/mdrA2– endosomes have an unusually high pH, whereas mdrA1–/mdrA2–/rtoA– endosomes have an almost normal pH. The ability of mdrA1/mdrA2 disruption to rescue the cell-type proportion, developmental defects, and endosomal pH defects caused by rtoA disruption, and the ability of rtoA disruption to exacerbate the endocytosis defects caused by mdrA1/mdrA2 disruption, suggest a genetic interaction between rtoA, mdrA1 and mdrA2.
Distinct changes in intranuclear lamin A/C organization during myoblast differentiation
Authors: Muralikrishna Bh. , Dhawan Jyotsna , Rangaraj Nandini , Parnaik Veena K.
Published: April 2021

Intranuclear lamin foci or speckles have been observed in various cell types. In order to explore the possibility of changes in internal lamin organization during muscle differentiation, we have examined the appearance of A-type lamin speckles that associate with RNA splicing factor speckles in C2C12 myoblasts and myotubes. Lamin speckles were observed in dividing myoblasts but disappeared early during the course of differentiation in postmitotic myocytes, and were absent in myotubes and muscle fibers. However, no changes were seen in the typical peripheral organization of lamins A/C or B1 or in RNA splicing factor speckles. Lamin speckles were also absent in quiescent myoblasts but reappeared as cells were reactivated to enter the cell cycle. These changes were not observed in other quiescent cell types. Immunoblot analysis indicated that the abundance and migration of lamins A and C was not altered in differentiated myoblasts. When myotube or quiescent myoblast nuclei were extracted with nucleases and detergent, a uniformly stained internal lamina was revealed, indicating that lamins A/C were antigenically masked in these cells, probably owing to structural reorganization of the lamina during differentiation or quiescence. Our results suggest that muscle cell differentiation is accompanied by regulated rearrangements in the organization of the A-type lamins.
Correction of delF508-CFTR activity with benzo(c)quinolizinium compounds through facilitation of its processing in cystic fibrosis airway cells
Authors: Dormer Robert L. , Dérand Renaud , McNeilly Ceinwen M. , Mettey Yvette , Bulteau-Pignoux Laurence , Métayé Thierry , Vierfond Jean-Michel , Gray Michael A. , Galietta Luis J. V. , Morris M. Rachel , Pereira Malcolm M. C. , Doull Iolo J. M. , Becq Frédéric , McPherson Margaret A.
Published: April 2021

A number of genetic diseases, including cystic fibrosis, have been identified as disorders of protein trafficking associated with retention of mutant protein within the endoplasmic reticulum. In the presence of the benzo(c)quinolizinium drugs, MPB-07 and its congener MPB-91, we show the activation of cystic fibrosis transmembrane conductance regulator (CFTR) delF508 channels in IB3-1 human cells, which express endogenous levels of delF508-CFTR. These drugs were without effect on the Ca2+-activated Cl– transport, whereas the swelling-activated Cl– transport was found altered in MPB-treated cells. Immunoprecipitation and in vitro phosphorylation shows a 20% increase of the band C form of delF508 after MPB treatment. We then investigated the effect of these drugs on the extent of mislocalisation of delF508-CFTR in native airway cells from cystic fibrosis patients. We first showed that delF508 CFTR was characteristically restricted to an endoplasmic reticulum location in approximately 80% of untreated cells from CF patients homozygous for the delF508-CFTR mutation. By contrast, 60-70% of cells from non-CF patients showed wild-type CFTR in an apical location. MPB-07 treatment caused dramatic relocation of delF508-CFTR to the apical region such that the majority of delF508/delF508 CF cells showed a similar CFTR location to that of wild-type. MPB-07 had no apparent effect on the distribution of wild-type CFTR, the apical membrane protein CD59 or the ER membrane Ca2+,Mg-ATPase. We also showed a similar pharmacological effect in nasal cells freshly isolated from a delF508/G551D CF patient. The results demonstrate selective redirection of a mutant membrane protein using cell-permeant small molecules of the benzo(c)quinolizinium family and provide a major advance towards development of a targetted drug treatment for cystic fibrosis and other disorders of protein trafficking.
Uroplakin Ia is the urothelial receptor for uropathogenicEscherichia coli: evidence from in vitro FimH binding
Authors: Zhou Ge , Mo Wen-Jun , Sebbel Peter , Min Guangwei , Neubert Thomas A. , Glockshuber Rudi , Wu Xue-Ru , Sun Tung-Tien , Kong Xiang-Peng
Published: April 2021

The binding of uropathogenic Escherichia coli to the urothelial surface is a crucial initial event for establishing urinary tract infection because it allows the bacteria to gain a foothold on the urothelial surface, thus preventing them from being removed by micturition. In addition, it triggers bacterial invasion as well as host urothelial defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium, a filamentous attachment apparatus, and its urothelial receptor. We have prepared a biotinylated, recombinant FimH-FimC adhesin:chaperone complex and used it to identify its mouse urothelial receptor. The FimH-FimC complex binds specifically to a single 24 kDa major mouse urothelial plaque protein, which we identified as uroplakin Ia by mass spectrometry, cDNA cloning and immunoreactivity. The terminal mannosyl moieties on Asn-169 of uroplakin Ia are responsible for FimH as well as concanavalin A binding. Although FimH binds to uroplakin Ia with only moderate strength (Kd ∼100 nM between pH 4 and 9), the binding between multiple fimbriae of a bacterium and the crystalline array of polymerized uroplakin receptors should achieve high avidity and stable bacterial attachment. The FimH-FimC complex binds preferentially to the mouse urothelial umbrella cells in a pattern similar to uroplakin staining. Our results indicate that the structurally related uroplakins Ia and Ib are glycosylated differently, that uroplakin Ia serves as the urothelial receptor for the type 1-fimbriated E. coli, and that the binding of uropathogenic bacteria to uroplakin Ia may play a key role in mediating the urothelial responses to bacterial attachment.
Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus
Authors: Yoshimura Shin-ichiro , Nakamura Nobuhiro , Barr Francis A. , Misumi Yoshio , Ikehara Yukio , Ohno Hiroshi , Sakaguchi Masao , Mihara Katsuyoshi
Published: April 2021

The targeting route of newly synthesized GM130 and GRASP65 to the Golgi apparatus was investigated by three different approaches. First, localization of pulse labeled GM130 and GRASP65 in normal rat kidney (NRK) cells was traced by subcellular fractionation followed by immunoprecipitation. Immediately after the pulse labeling, GM130 and GRASP65 were found in the Golgi but not in the endoplasmic reticulum (ER) membrane fractions, whereas a control Golgi membrane protein was still found in the ER membrane fractions. Second, epitope tagged GM130 and GRASP65 were expressed in NRK cells by plasmid microinjection into the nuclei and their localization was analyzed by immunofluorescence. When ER to Golgi transport was inhibited by prior microinjection of a GTP-restricted mutant of Sar1 protein into the cytosol, the expressed GM130 and GRASP65 showed clear Golgi localization. Last, binding of GM130 and GRASP65 to the membranes was analyzed in vitro. In vitro synthesized GM130 and GRASP65 specifically bound to purified Golgi membranes but not to microsomal membranes. The bound GM130 and GRASP65 were found to form a complex with pre-existing counterparts on the Golgi membrane. These results strongly suggested that GM130 and GRASP65 are directly targeted to the Golgi membrane without initial assembly on the ER and subsequent vesicular transport to the Golgi apparatus.
Nucleolus-associated telomere clustering and pairing precede meiotic chromosome synapsis in Arabidopsis thaliana
Authors: Armstrong Susan J. , Franklin F. Christopher H. , Jones Gareth H.
Published: April 2021

The intranuclear arrangements of centromeres and telomeres during meiotic interphase and early prophase I of meiosis in Arabidopsis thaliana were analysed by fluorescent in situ hybridisation to spread pollen mother cells and embryo-sac mother cells. Meiocyte identification, staging and progression were established by spreading and sectioning techniques, including various staining procedures and bromodeoxyuridine labeling of replicating DNA.

Centromere regions of Arabidopsis are unpaired, widely dispersed and peripherally located in nuclei during meiotic interphase, and they remain unpaired and unassociated throughout leptotene. Eventually they associate pairwise during zygotene, as part of the nucleus-wide synapsis of homologous chromosomes.

Telomeres, by contrast, show a persistent association with the nucleolus throughout meiotic interphase. Variation in telomere signal number indicates that telomeres undergo pairing during this interval, preceding the onset of general chromosome synapsis. During leptotene the paired telomeres lose their association with the nucleolus and become widely dispersed. As the chromosomes synapse during zygotene, the telomeres reveal a loose clustering within one hemisphere, which may represent a degenerate or relic bouquet configuration. We propose that in Arabidopsis the classical leptotene/zygotene bouquet is absent and is replaced functionally by nucleolus-associated telomere clustering.
Intranuclear endoplasmic reticulum induced by Nopp140 mimics the nucleolar channel system of human endometrium
Authors: Isaac Cynthia , Pollard Jeffrey W. , Meier U. Thomas
Published: April 2021

Exogenous expression of the characteristic repeat domain of the nucleolar chaperone Nopp140 induces the formation of intranuclear structures, termed R-rings. Here, the R-rings are identified as extensive stacks of membrane cisternae in the otherwise membrane-free nucleus. They consist of bona fide endoplasmic reticulum (ER) containing integral membrane proteins of the smooth and rough ER. Although lacking nuclear pore complexes and lamina, the R-rings derive specifically from the inner nuclear membrane. These findings are consistent with the idea that all transmembrane proteins synthesized in the ER and the outer nuclear membrane can freely diffuse through the pore membrane domain into the inner membrane. Uniquely, the soluble transfected Nopp140 is directly involved in the generation of these membrane stacks as it localizes to the electron dense matrix in which they are embedded. The only well-documented example of intranuclear membrane proliferation is the nucleolar channel system of the postovulation human endometrium. The transient emergence of the nucleolar channel system correlates precisely with the readiness of the endometrium for the implantation of the fertilized egg. The nucleolar channel system exhibits an ultrastructure that is indistinguishable from R-rings, and nuclei of human endometrium harbor Nopp140 and ER marker containing structures. Therefore, the nucleolar channel system appears to be identical to the R-rings, suggesting a role for Nopp140 in human reproduction.
Chlamydomonas reinhardtiiproduces a profilin with unusual biochemical properties
Authors: Kovar David R. , Yang Pinfen , Sale Winfield S. , Drobak Bjørn K. , Staiger Christopher J.
Published: April 2021

We report the characterization of a profilin orthologue from Chlamydomonas reinhardtii. CrPRF, probably the only profilin isoform, is present in both the cell body and flagella. Examination of vegetative and gametic cells by immunofluorescence microscopy using multiple fixation procedures also revealed enrichment of CrPRF at the anterior of the cell near the base of flagella and near the base of the fertilization tubule in mating type plus gametes. Purified, recombinant CrPRF binds to actin with a Kd value ∼10–7 and displaces nuclei in a live cell ‘nuclear displacement’ assay, consistent with profilin’s ability to bind G-actin in vivo. However, when compared with other profilin isoforms, CrPRF has a relatively low affinity for poly-L-proline and for phosphatidylinositol (4,5) bisphosphate micelles. Furthermore, and surprisingly, CrPRF inhibits exchange of adenine nucleotide on G-actin in a manner similar to human ADF or DNase I. Thus, we postulate that a primary role for CrPRF is to sequester actin in Chlamydomonas. The unusual biochemical properties of CrPRF offer a new opportunity to distinguish specific functions for profilin isoforms.
Cytoskeletal control of fibroblast length: experiments with linear strips of substrate
Authors: Levina Elina M. , Kharitonova Margarita A. , Rovensky Yuri A. , Vasiliev Jury M.
Published: April 2021

In order to understand the factors determining the length of fibroblasts, three cell lines (mouse embryonic fibroblasts plus human fibroblast lines AGO 1523 and M19) were cultivated on the usual planar substrate (glass) and on specially prepared narrow linear strips of the same substrate, where the cells could spread only linearly. Morphometric measurements showed that the average length of cells of each type on the ‘unidimensional’ strips was no different from that on the usual ‘bidimensional’ substrate. The addition of colcemid significantly decreased cell length on both substrates, whereas cytochalasin D increased the length. We concluded that fibroblasts have an intracellular mechanism maintaining a relatively constant average cell length. This mechanism may involve the dynamic balance of centripetal and centrifugal forces developed by two cytoskeletal systems: the microtubules and the actin-myosin cortex. Three epitheliocyte cell lines (rat IAR2, canine MDCK and bovine FBT) were tested but, in contrast to fibroblasts, they did not maintain similar cell lengths on the usual substrate and on the linear strips, suggesting that control of length is cell-type-specific.
Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells
Authors: Taylor Stephen S. , Hussein Deema , Wang Yunmei , Elderkin Sarah , Morrow Christopher J.
Published: April 2021

BUB1 is a budding yeast gene required to ensure that progression through mitosis is coupled to correct spindle assembly. Two related human protein kinases, Bub1 and BubR1, both localise to kinetochores during mitosis, suggesting that they play a role in delaying anaphase until all chromosomes achieve correct, bipolar attachment to the spindle. However, how the activities of Bub1 and BubR1 are regulated by spindle events and how their activities regulate downstream cell cycle events is not known.

To investigate how spindle events regulate Bub1 and BubR1, we characterised their relative localisations during mitosis in the presence and absence of microtubule toxins. In prometaphase cells, both kinases colocalise to the same domain of the kinetochore. However, whereas the localisation of BubR1 at sister kinetochores is symmetrical, localisation of Bub1 is often asymmetrical. This asymmetry is dependent on microtubule attachment, and the kinetochore exhibiting weaker Bub1 staining is typically closer to the nearest spindle pole. In addition, a 30 minute nocodazole treatment dramatically increases the amount of Bub1 localising to kinetochores but has little effect on BubR1. Furthermore, Bub1 levels increase at metaphase kinetochores following loss of tension caused by taxol treatment. Thus, these observations suggest that Bub1 localisation is sensitive to changes in both tension and microtubule attachment.

Consistent with this, we also show that Bub1 is rapidly phosphorylated following brief treatments with nocodazole or taxol. In contrast, BubR1 is phosphorylated in the absence of microtubule toxins, and spindle damage has little additional effect. Although these observations indicate that Bub1 and BubR1 respond differently to spindle dynamics, they are part of a common complex during mitosis. We suggest therefore that Bub1 and BubR1 may integrate different ‘spindle assembly signals’ into a single signal which can then be interpreted by downstream cell cycle regulators.
Regulation of Golgi structure and function by ARF-like protein 1 (Arl1)
Authors: Lu Lei , Horstmann Heinz , Ng Cheepeng , Hong Wanjin
Published: April 2021

Arl1 is a member of the ARF-like protein (Arl) subfamily of small GTPases. Nothing is known about the function of Arl1 except for the fact that it is essential for normal development in Drosophila and that it is associated with the Golgi apparatus. In this study, we first demonstrate that Arl1 is enriched at the trans side of the Golgi, marked by AP-1. Association of Arl1 with the Golgi is saturable in intact cells and depends on N-terminal myristoylation. Over-expression of Arl1(T31N), which is expected to be restricted to the GDP-bound form and thus function as a dominant-negative mutant, causes the disappearance of the Golgi apparatus (marked by Golgi SNARE GS28), suggesting that Arl1 is necessary for maintaining normal Golgi structure. Overexpression of Arl1(Q71L), a mutant restricted primarily to the activated GTP-bound form, causes an expansion of the Golgi apparatus with massive and stable Golgi association of COPI and AP-1 coats. Interestingly, Golgi ARFs also become stably associated with the expanded Golgi. Transport of the envelope protein of vesicular stomatitis virus (VSV-G) along the secretory pathway is arrested at the expanded Golgi upon expression of Arl1(Q71L). The structure of stacked cisternae of the Golgi is disrupted in cells expressing Arl1(Q71L), resulting in the transformation of the Golgi into an extensive vesicule-tubule network. In addition, the GTP form of Arl1 interacts with arfaptin-2/POR1 but not GGA1, both of which interact with GTP-restricted ARF1, suggesting that Arl1 and ARF1 share some common effectors in regulating cellular events. On the basis of these observations, we propose that one of the mechanisms for the cell to regulate the structure and function of the Golgi apparatus is through the action of Arl1.
Identification of essential genes in cultured mammalian cells using small interfering RNAs
Authors: Harborth Jens , Elbashir Sayda M. , Bechert Kim , Tuschl Thomas , Weber Klaus
Published: April 2021

We report the first RNAi-induced phenotypes in mammalian cultured cells using RNA interference mediated by duplexes of 21-nt RNAs. The 21 gene products studied have different functions and subcellular localizations. Knockdown experiments monitored by immunofluorescence and immunoblotting show that even major cellular proteins such as actin and vimentin can be silenced efficiently. Genes were classified as essential or nonessential depending on impaired cell growth after RNA silencing. Phenotypes also involved altered cell morphology and aberrant mitotic arrest. Among the essential genes identified by RNAi for which such information was previously not available are lamin B1, lamin B2, NUP153, GAS41, ARC21, cytoplasmic dynein, the protein kinase cdk1 and both β- and γ-actin. Newly defined nonessential genes are emerin and zyxin. Several genes previously characterized by other methods such as knockout of murine genes are included as internal controls and gave identical results when RNAi was used. In the case of two nonessential genes (lamin A/C and zyxin) RNAi provides a recognizable phenotype.

Our results complete the characterization of the mammalian nuclear lamins. While lamins A/C appear as nonessential proteins in the mouse embryo and in RNAi treated cultured cells, the two other lamins, B1 and B2, are now identified as essential proteins. Interestingly the inner nuclear membrane protein emerin, thought to be a ligand of lamin A/C, is also a nonessential protein in tissue culture cells.
A common molecular machinery for exocytosis and the ‘kiss-and-run’ mechanism in chromaffin cells is controlled by phosphorylation
Authors: Henkel Andreas W. , Kang Guoxin , Kornhuber Johannes
Published: April 2021

Exocytosis and ‘kiss-and-run’ secretion coexist in chromaffin cells. Our findings suggest that these mechanisms are closely related, based on their common molecular machinery. Here we present a model that describes how chromaffin cells regulate catecholamine release by switching the mode of secretion between the two pathways, a process controlled by phosphorylation. Stimulation-dependent vesicle-plasma membrane interactions in chromaffin cells were analysed by simultaneous ‘on-cell’ capacitance and conductance measurements, a technique that allows the monitoring of single vesicles. Capacitance steps represent fusions of large dense-core vesicles with the plasma membrane, whereas capacitance flickers correspond to transient connections of the vesicle lumen with the extracellular space. All these events require the presence of extracellular calcium in millimolar concentrations. ‘Kiss-and-run’ type of release is enhanced by the kinase inhibitor staurosporine, which suggests that this secretion mode is regulated by protein phosphorylation. We also observed capacitance bursts, which most probably represent ‘hot spots’ of secretion and we found that ‘kiss-and-run’ is the prevalent mechanism during these episodes. The significance of ‘kiss-and run’ for neurohormone release is even higher at physiological temperature, because up to half of all secretion events are mediated by this mechanism.
Localized activation of bending in proximal, medial and distal regions of sea-urchin sperm flagella
Authors: Brokaw C. J. , Gibbons I. R.
Published: April 2021

Spermatozoa from the sea urchin, Colobocentrotus atratus, were partially demembranated by extraction with solutions containing Triton X-100 at a concentration which was insufficient to solubilize the membranes completely. The resulting suspension was a mixture containing some spermatozoa in which a proximal, medial, or distal portion of the flagellum was membrane covered, while the remaining portion was naked axoneme. In reactivating solutions containing 12 μ M ATP, only the naked portions of the flagellum became motile. In reactivating solutions containing 0·8 mM ADP, the membrane-covered regions became motile and beat at 6–10 beats/s, while the naked regions remained immobile, or beat very slowly at about 0·3 beat/s. Activation of membrane-covered regions in ADP solutions probably results from the membrane restricting the diffusion of ATP which is formed from ADP by the axonemal adenylate kinase. The results indicate that any region of the flagellum has the capacity for autonomous beating, and that special properties of the basal end of the flagellum are not required for bend initiation. How ever, the beating of different regions of the flagellum is not completely independent, for in a fair number of spermatozoa the beating of the distal, membrane-covered region in 0·8 mM ADP was intermittent, and was turned on and off in phase with the much slower bending cycle in the proximal region of naked axoneme.
Intermediate cells of the pancreas: III. selective autophagy and destruction of β-granules in intermediate cells of the rat pancreas induced by alloxan and streptozotocin
Authors: Melmed R. N. , Benitez Carol J. , Holt S. J.
Published: April 2021

In addition to causing necrosis ofβcells, diabetogenic doses of alloxan or streptozotocin cause selective autophagy and destruction of β-granules in intermediate cells in the rat pancreas, thus providing evidence that these granules and those in the β-cells of the islet are identical. In acinar-β cells the autophagic vacuoles also contain small mitochondria similar to those that occur in islet endocrine cells. Apart from these effects, the cells remain structurally intact. These observations suggest that alloxan exerts a direct effect on the β-components of the β-granule-containing intermediate cells.

The destruction of the β-granules in the acinar-β cell is sometimes accompanied by the appearance of α-granules in the same cells, thus presumably reflecting the increased demand for glucagon that occurs in experimental diabetes. Although intermediate cells containing α-granules are very uncommon in the normal rat pancreas, acinar-αand α-acinar cells occurred much more frequently after alloxan treatment.

The presence of intermediate cells of the acinar-β type in normal rats and the observed change of acinar-β to acinar-α cells, or from α- to α-acinar cells after alloxan, indicates that some pancreatic cells possess a variable functional potentiality. The appearance of these cells after alloxan presumably reflects the altered metabolic state of the animal, and is not a manifestation of the transformation of one cell type into another.
Cathepsin D in cartilage: The immunohistochemical demonstration of extracellular enzyme in normal and pathological conditions
Authors: Poole A. R. , Hembry R. M. , Dingle J. T.
Published: April 2021

Extracellular cathepsin D has been demonstrated immunohistochemically in cultured chicken and rabbit cartilage. Enzyme released from viable cells was captured by specific anti-cathepsin D antibody to form extracellular immunoprecipitates which were demonstrated with fluorescein-labelled antibodies. In unfixed frozen sections these immunoprecipitates could be dissolved in antigen excess, and closely resembled those observed in immunoprecipitin lines. Extracellular cathepsin D was detected in the perichondria of normal chick cartilaginous limb-bones and, to a lesser degree, beneath them in the epiphyseal cartilage. In cultured rabbit ear cartilage, cathepsin D was detected around chondroblasts and some peripherally situated chondrocytes. In chicken and rabbit cartilage exposed to supra-physiological levels of retinol, much extracellular enzyme was detected which had been released from chondrocytes of hypertrophic and epiphyseal chick cartilage and rabbit ear cartilage.
An Examination Into The Phenomenon Of Granulation, In Mu Particles Of Stock 540 Paramecium Aurelia
Authors: Franklin J. , Baker N. O.
Published: April 2021

Mu particles in paramecia incubated at 37 °C granulate. The treatment causes the loss of mu and granulation is a prerequisite for loss. The granules form along the length of the mu, at intervals of about 1·50 μm. Each granule has a diameter of about 0·5 μm and consists of a number of ribosome-like particles which have the same dimensions as 70s ribosomes.
Correlation between resistance to actinomycin d, karyology, agglutination by concanavalin a and tumorigenicity in chinese hamster hybrid cells
Authors: Imbert I. , Barra Y. , Berebbi M.
Published: April 2021

Subclones isolated from a Chinese hamster hybrid line, derived from fusion of an actinomycin D-resistant and an actinomycin D-sensitive strain, were studied with respect to their resistance to actinomycin. D, karyology, transplantability and agglutination by concanavalin A. Statistical analysis of the results allowed the establishment of a classification of the strains based on increasing resistance to actinomycin D. There appeared to be an inverse correlation between actinomycin D-resistance and tumorigenicity and a positive correlation between this resistance and the presence of a marker chromosome.
Perturbation of mammalian cell division: II studies on the isolation and characterization of human mini segregant cells
Authors: Schor S. L. , Johnson R. T. , Mullinger A. M.
Published: April 2021

A method is described for the isolation, according to size, of mini segregants produced by the abnormal cleavage of reversibly arrested mitotic HeLa cells. Many of these mini segregants contain small amounts of DNA, as judged by Feulgen staining and chromosome analysis. After fusion with mitotic HeLa cells, the interphase chromosomes of the mini segregants are seen as either monovalent or bivalent prematurely condensed chromosomes (PCC), some of which are damaged. A proportion of isolated mini segregants synthesize DNA, RNA and protein. Fusion of mini segregants with interphase HeLa cells gives rise to cells with ‘hybrid’ karyotypes.