bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of size regulation in mouse embryo aggregates
Authors: Lewis N. E. , Rossant J.
Published: April 2021

A detailed comparison of the postimplantation development of normal and double-sized mouse embryos, produced by aggregating two 8-cell stage eggs, revealed that size regulation occurred in the double embryos between 5 days, 16 h post coitum (p.c.) and 6 days, 16 h p.c. Size regulation occurred simultaneously in all tissues, suggesting that a single regulatory mechanism may control size in the early embryo. Size regulation appeared to be brought about by alteration in cell cycle length. There was no obvious increase in cell death in the double embryos nor an increase in the non-dividing cell population. However, colcemid treatment revealed a significant difference in mitotic index between double and control embryos over the period of size regulation. Control embryos showed a proliferative burst around 6 days, 8 h p.c. which did not occur in the double embryos. It is not yet clear whether this control of proliferative activity in double embryos is exerted by the embryo itself or by the uterine environment.

Histological analysis also suggested that proamniotic cavity formation, which occurs before size regulation, was dependent on total cell number and not on the number of cell cycles undergone since fertilization. Proamniotic cavity formation was observed to occur at different times but at similar cell numbers in double, control and half embryos.
Comparative Investigations of the Action of Two Nitrogen Mustard Derivatives on the Early Stages of Development of Chick Embryos
Authors: Jurand A.
Published: April 2021

Cytotoxic compounds act by combining with the biochemical constituents of cells. Because of the complexity of living matter, the cytotoxic activity is highly complicated in nature and is therefore far from being thoroughly understood. In order to analyse the cytotoxicity of any chemical compound, many biological variables concerned in determining the mode of action of the compound and its selectivity for any particular range of cells have to be taken into account (Danielli, 1952,1954).
The Growth of the Locust Embryo
Authors: Salzen E. A.
Published: April 2021

Three measures of the growth of the embryo of Locusta migratoria migratorioides (R. & F.) were determined from the third to the twelfth days of development at 30° C. These measures were the dry weight, reduced weight, and number of nuclei. Measurements of the density of the embryo were also made and used with the R.W. to calculate the wet weight. The same measurements of the intact egg were taken throughout development. The growth in dry weight of the locust embryo from the third to twelfth days of development appeared closely comparable with that of vertebrate embryos such as the chick embryo and could be described by the usual double logarithmic relation with growth constants of closely comparable values. Increase in R.W. and wet weight could be similarly described from the third to ninth days after which the rate of increase fell away more rapidly than expected. Increase in nuclear number occurred at a higher rate than any of the other measures up to the ninth day after which no further increase above a total of about two million nuclei could be detected. As in some vertebrate embryos, cell multiplication seemed not to parallel growth in weight. The implications of the differences in rates of increase of these measures were considered. From the third to ninth days the embryo was approximately 90 per cent, water, the density of the dry material may have increased and the dry weight per nucleus decreased rapidly to a constant value. After the ninth day there appeared to be a fall in water content and dry density and an increase in the dry weight/nuclear number ratio. The density of the wet embryo increased steadily after the sixth day. The total loss in dry weight of the egg due to respiration was about 20 per cent., an amount typical of many insect eggs. The eggs increased in weight by about 120 per cent., chiefly during the fourth and fifth days. The percentage water content rose from about 50 per cent, to about 80 per cent. Changes in the density of the eggs reflected perfectly the changes in water content. The R.W. of the eggs was constant except for a rise of about 40 μ g. during the fourth and fifth days. It was suggested that this could be accounted for by the presence in the chorion of about 0 04 /xl. of air which was displaced when water uptake took place after the third day. This would also have accounted for the very small difference in the measured changes in density compared with those calculated from the water intake. A constant R.W. and a large decrease in dry weight imply that the density of the dry material of the eggs must have increased at the same rate as the respiratory loss in dry weight. Combustion of fat might account for this.
Stimulating Action of Tissue Extracts on Regenerative Processes
Authors: Tumanishvili G. D.
Published: April 2021

By means of systematic injections of tissue extracts one may obtain stimulation of regeneration of muscles (in frogs) and liver (in frogs, rabbits, guinea-pigs). Such extracts, however, apparently only influence homologous tissue. The influence of extracts does not show any class specificity. On the con-trary, the stimulation of regeneration of frog muscle and liver is more effectively produced by corresponding extracts of hen tissues than by those of the frog. Aqueous extracts are as active as those obtained with isotonic saline. Heating to 60° C. for 5 minutes inactivates the extract. The analysis of our data and of those published elsewhere leads to the conclusion that the stimulating agent is a water-soluble protein fraction, or that it is some substance (e.g. RNA) forming a rather strong complex with this fraction.
Differentiation of allantoic endoderm implanted into the presumptive digestive area in avian embryos. A study with organ-specific antigens
Authors: Yasugi Sadao
Published: April 2021

Quail allantoic endoderm was implanted into the presumptive digestive-tract area of chick embryos, and the differentiation of the endoderm was examined morphologically and immunocytochemically with antisera against pepsinogens and sucrase. The allantoic endoderm was incorporated into the host digestive organs. It often became continuous with the host endoderm and formed a chimaeric digestive-tract epithelium. It differentiated morphologically into the epithelium of the digestive organ into which it was incorporated, showing the morphological inductive ability in situ of the digestive-tract mesenchyme against the allantoic endoderm. However, the allantoic endoderm did not produce pepsinogens even when it was incorporated into the host proventricular mesenchyme and formed well-developed proventricular glands. This result indicates that the heterotypic morphogenesis of the allantoic endoderm is not necessarily accompanied by the heterotypic cytodifferentiation. In contrast, the anti-sucrase antiserum-reactive cells often differentiated in the allantoic endoderm incorporated into not only the intestine but also other organs. This confirmed our previous observation that the allantoic endoderm has a tendency to differentiate into the intestinal epithelium in the heterologous environment.
Genetic analysis of developmental mechanisms in hydra
Authors: Takano Jun , Sugiyama Tsutomu
Published: April 2021

Chimaeric hydra strains were produced from a normal strain (105) and a naturally-occurring mutant strain (L4) which has a large polyp size, a low budding rate and a high head-inhibition potential. Various properties of the chimaeras were then examined and compared to those of the two parental strains.

Hydra tissue consists of three cell lineages: the ectodermal epithelial, the endodermal epithelial and the interstitial cell lineages. Using the methods recently developed by Marcum & Campbell (1978b) and by Wanek & Campbell (1982), six chimaeric strains were produced which contained six different combinations of the three cell lineages from 105 and L4.

Evidence obtained from the comparison of the chimaeras and their parental strains indicates that the ectodermal epithelial cell lineage in L4 is primarily responsible for the large polyp size and the low budding rate of this strain, whereas the endodermal epithelial cell lineage is largely, and the interstitial cell lineage is also partially, responsible for the high head-inhibition potential in L4. This suggests that the mechanisms determining the occurrence and location of bud formation and the mechanisms determining the inhibition potential levels are not related to each other (cf. Takano & Sugiyama, 1983; Bode & Bode, 1983). Evidence was also obtained which suggests that the levels of the head-activation and head-inhibition potentials in the chimaeras are determined independent of each other, apparently without the cross-catalytic relationship between them assumed in the Gierer-Meinhardt model (Gierer & Meinhardt, 1972; Meinhardt & Gierer, 1974).
Ontogeny of the granulocyte/macrophage progenitor cell (GM-CFC) pools in the beagle
Authors: Nothdurft Wilhelm , Braasch Eckehard , Calvo Wenceslao , Prümmer Otto , Carbonell Felix , Grilli Gualtiero , Fliedner Theodor M.
Published: April 2021

The pattern of development of the granulocyte/macrophage progenitor cell (GM-CFC) pools in the course of canine ontogeny was studied by means of the agar culture technique. Colony formation was stimulated by colony stimulating activity (CSA) in serum from lethally irradiated dogs in combination with erythrocyte-depleted peripheral blood leukocytes from normal adult dogs. The colonies thus obtained in cultures from the different organs were in general large (estimated maximum 50000 cells) and consisted predominantly of mononucleated macrophages, suggesting that, in these studies, a progenitor cell with high proliferative potential (HPP-CFC) has been monitored.

In the yolk sac, a transitory GM-CFC pool became established between day 23 and day 48 of gestation, reaching maximum numbers of approximately 41 × 103 per organ on days 36β7. At the same time the GM-CFC concentration in blood collected from the heart also reached a maximum of about 31 × 103/ml, indicating its carrier function for the migration of GM-CFC. In the liver a quasi-exponential increase in the GM-CFC numbers took place between days 36β7 and days 57 to 59 when a total of about 15·2 × 106 was found but thereafter and up to day 4 post partum the GM-CFC numbers decreased by almost two orders of magnitude. A continuous increase in the GM-CFC numbers was found in the spleen between day 42 of gestation and day 4 post partum when a maximum of 5·l × 106 to 8·7 × 106 was reached. In contrast to the GM-CFC numbers in the liver, the splenic GM-CFC dropped only by 50 % of peak values when the dogs reached adulthood. The bone marrow always had the highest incidence of GM-CFC, the concentration per 106 cells being 18·7 × 103/106 cells on days 45/46, the earliest time point at which cultures could be set up. The absolute GM-CFC numbers in the two femora increased continuously between days 45/46 and day 4 post partum in parallel with the growth of the bones. In the thymus a relatively small population of GM-CFC developed between days 42 and 48 of gestation that was kept quite constant at average numbers between 13 × 103 and 30 × 103 up to day 4 post partum.
Changes in actin distribution during fertilization of the mouse egg
Authors: Maro B. , Johnson M. H. , Pickering S. J. , Flach G.
Published: April 2021

Unfertilized mouse oocytes and eggs 1–8 h after fertilization in vitro were examined at the light microscope level for structural changes, distribution of actin (as assessed by both antiactin antibodies and NBD-phallicidin), surface binding of concanavalin A (Con A) and chromosomal distribution and condensation. The influence of cytochalasin D on these events was also assessed. Changes in actin distribution were associated with rotation of the second anaphase spindle, formation of the second polar body, the events following incorporation of the sperm nucleus, and formation and migration of the pronuclei. Cytochalasin D prevented spindle rotation, polar body formation, pronuclear migration and the restoration of Con A binding over the site of sperm entry and the site of polar body formation, but did not affect sperm fusion and entry, the loss of Con A binding at the site of sperm entry and pronuclear formation.
The differentiation in vitro of the neural crest cells of the mouse embryo
Authors: Ito Kazuo , Takeuchi Takuji
Published: April 2021

A culture method for neural crest cells of mouse embryo is described. Trunk neural tubes were dissected from 9-day mouse embryos and explanted in culture dishes. The developmental potential of mouse neural crest in vitro was shown to be essentially similar to that of avian neural crest. In the mouse, however, melanocytes always appeared in association with the epithelial sheet close to the explant. Neural crest cells surrounding the epithelial sheet, which probably migrated from the neural tubes in the early culture phase, never differentiated into melanocytes. The bimodal behaviour of mouse crest cells seems to be due to the heterogenous potency of the crest cells and the interaction of these cells with the surrounding microenvironment. This culture system is well suited for various experiments including the analysis of gene control on the differentiation of neural crest cells.
Effects of cytochalasin B on the formation of previllous ridges and the appearance of long microvillous-like processes in the organ culture system of chick embryonic intestine
Authors: Noda Setsuko
Published: April 2021

Between 8 and 12 days of incubation the embryonic duodenum serially constructs with relative regularity the previllous ridges upon which the definitive villi later form. The effects of cytochalasin B (CB) on the formation of these previllous ridges of the duodena of developing chick embryos were studied, varying the concentrations and exposure time of CB in the organ culture system. The results were as follows:- (1) CB inhibited the formation of new previllous ridges from the epithelial cell sheets of 8-to 11-day-old embryonic duodena at a cultured time of 24 h. (2) CB treatment blocked or delayed cytokinesis of the epithelial cells and the production of many long microvillous-like processes (long processes) from the surface of the epithelial cells. (3) These long processes elicited by CB contained actin filaments and their appearance was influenced by the developmental stages of embryos and local parts of epithelial cells. (4) With 11-day embryonic duodena, induction of long processes by CB was observed at various concentrations (1 μg/ml–16 μg/ml) and even after short exposure of 15 min. (5) Cytochalasin D (CD) and colchicine were used and long processes were induced by CD but not by colchicine itself. The appearance of long processes depended on the experimental concentration of CB, CD and colchicine.

In normal developments, such long processes appeared and disappeared within a confined area during the formation of previllous ridges (Noda, 1981). This study seemed to provide experimental support for the previous reported suggestion that the long processes might be one of the important factors in the formation of the previllous ridges of chick embryonic duodena.
Histospecific acetylcholinesterase development in the presumptive muscle cells isolated from 16-cell-stage ascidian embryos with respect to the number of DNA replications
Authors: Mita-Miyazawa Izumi , Ikegami Susumu , Satoh Noriyuki
Published: April 2021

The presumptive muscle cells (B5.1 blastomeres) were isolated from 16-cell-stage embryos of the ascidian, Ciona intestinalis. The isolated cells were allowed to divide either twice or three times thereafter. Then further divisions of the cells were continuously inhibited by a simultaneous treatment with aphidicolin (a specific inhibitor of DNA synthesis) and cytochalasin B (an inhibitor of cytokinesis). When development of muscle-specific acetylcholinesterase in these division-arrested progeny cells of B5.1 blastomeres was examined histochemically, the B5.1 blastomeres which had been allowed two further divisions did not produce any detectable acetylcholinesterase activity. Whereas those which had been allowed three further divisions showed the tissue-specific enzyme activity. These results provide further evidence for the presence of a quantal DNA replication cycle for the tissue-specific enzyme development, which is qualitatively different from the other DNA replication cycles.
Influence of Temperature on Rate of Regeneration in the Time-graded Regeneration Field in Planarians
Authors: Agnes  , Brøndsted H. V.
Published: April 2021

The aim of the experiments was to see how different levels in the time-graded head-regeneration field of two species, differing strongly as to the characteristics of the field, might behave at different temperatures. As was expected, regeneration was retarded for all levels by lowering the temperature. Regeneration of heads was retarded relatively more strongly at posterior levels of the field. Some characteristic differences in regeneration rates of the two species were found, leading to certain suggestions as to the possible nature of the field. By lowering the temperature regeneration may be separated into two main components : formation of the blastema and differentiation of the cells. Formation of the blastema is not seriously impeded at temperatures below 5–6° C., whereas the differentiation processes are nearly brought to a stop at these low temperatures. Doubt is therefore cast on the hypothesis of an adaptive value of regenera-tion in view of the fact that several planarian species actually live and reproduce at temperatures well below those necessary for regeneration.
The Association of Mucopolysaccharides with Morphogenesis of the Palate and Other Structures in Mouse Embryos
Authors: Walker Bruce E.
Published: April 2021

Acid mucopolysaccharides have been postulated to occur in the palatine shelves of mouse embryos on the basis of metachromatic staining with toluidine blue (Walker & Fraser, 1956), and this idea has since been supported by results from radioautography with S35 (Larsson, Bostrom, & Carlsoo, 1958). It was not known which structural component of the tissue contained this substance, but, since aldehyde fuchsin-positive material existed in the same area, the possibility was raised that the component was a network of elastic fibres (Walker & Fraser, 1956). The first purpose of the present paper is to describe further histochemical and radioautographic investigations of this problem, supplemented by electron microscopic observations of palatine shelf tissue.
La Métachromasie au bleu de toluidine dans l’œuf d’Artemia salina
Authors: Fautrez par J. , Fautrez-Firlefyn N.
Published: April 2021

Toluidine blue staining of fixed eggs of Artemia salina reveals during vitellogenesis, maturation, and segmentation the presence of metachromatic granules. These granules originate in certain yolk platelets. Scattered throughout the yolk, they show a series of progressive transformations while migrating into the perinuclear plasm, which they reach at the time of copulation of the pronuclei. During the cleavage divisions they are principally located around the asters. New elements continue to be formed within the yolk until the end of cleavage, when metachromatic elements disappear from the egg. Though they have obviously something to do with the rapid succession of mitoses during cleavage, their formation continues nevertheless in virgin females whose eggs are blocked in metaphase of the first maturation division.
The Importance of Mesenchymal Factors in the Differentiation of Chick Epidermis
Authors: Mcloughlin C. B.
Published: April 2021

The normal differentiation in vitro of whole skin from the limb-bud of the 5-day chick embryo is described briefly. Epidermis isolated by tryptic digestion from the 5-day limb-bud and cultivated alone in vitro shows very few mitoses and keratinizes completely within 10 days. Isolated epidermis grown in vitro in a medium containing excess vitamin A fails to keratinize, secretes mucus and sometimes becomes ciliated; mitoses are rare. Isolated epidermis explanted in contact with cell-free intercellular material from the mesenchyme becomes oriented towards it and arranged around it in an epithelial manner. These findings indicate that while the embryonic epidermis can differentiate in the absence of mesenchyme, contact with a basement membrane and the proximity of mesenchyme and its intercellular material are required for the normal growth and functioning of epithelial cells.
Recherche sur la différenciation protéique au cours du développement des Amphibiens
Authors: Denis par H.
Published: April 2021

Saline extracts of embryos of stages 0–37 (Pleurodeles waltii) were subjected to electrophoresis on cellulose acetate. Various embryonic tissues were studied in the same way. A new band appears in the electrophoretic pattern of whole embryos at the onset of gastrulation. Only minor changes can be detected until stage 31. Thereafter, the electrophoretic pattern becomes rapidly more complicated. From stage 0 to stage 31, there is a slow increase of the overall amount of extractable protein per embryo. After stage 31, soluble proteins appear at an increased rate. This acceleration is probably due to the starting of blood circulation. The various embryonic tissues already have a different protein constitution at the beginning of gastrulation. The proteins seem to be distributed in the early gastrula according to a dorso-ventral gradient. Competent ectoderm and neurulating ectoderm contain particular protein bands. From the second part of gastrulation onwards, the protein content of ventral ectoderm is different from that of neural ectoderm. Every embryonic tissue (except endoderm) acquires new protein bands during neurulation. These bands are different in each tissue. The embryo probably has two types of yolk. These two types are not digested at the same time but successively. The first type is probably located in the axial organs. The second type is contained in the endoderm and seems to be digested only after stage 31.
The Action of Various Agents upon the Rabbit Embryo
Authors: Adams C. E. , Hay Mary F. , Lutwak-Mann Cecilia
Published: April 2021

A study was made of the action upon the rabbit embryo of various agents administered to pregnant animals in doses which did not seriously upset maternal health. The pre-implantation blastocyst, examined as a flat mount, was the principal object of study, but observations were also made on ovulation, fertilization, cleavage, nidation, and subsequent phases of pregnancy. To facilitate comparison between embryos from normal and treated animals 5-to 7-day-old blastocysts from untreated animals, grouped according to size, were classified into 6 developmental stages, depending upon the degree of maturity of the embryonic disc. Additional procedures which served to evaluate the condition of blastocysts and their uterine environment were: mitotic counts made in the abembryonic part of the trophoblast, chemical determinations of certain characteristic constituents of blastocyst fluid, and measurement of carbonic anhydrase activity in the endometrium. . The experimental agents comprised hormones, colchicine derivatives, several polyfunctional alkylating compounds, metabolic analogues, a sulphonamide inhibitor of carbonic anhydrase, and substances endowed with teratogenic, hepatotoxic, radiation-protective, and hypoglycaemia-inducing properties; total body exposure to X-rays was also investigated. Results obtained at various stages of pregnancy with the different treatments are described and discussed; several results relating to blastocysts are illustrated by photomicrographs. Among significant general findings emerging from the study are: the speed with which exogenous agents are transmitted to embryos even before uterine attachment; differential drug sensitivity, frequently evident in the pre-implantation blastocysts, as between the embryonic disc and trophoblast, the former usually being more susceptible; and variability in response, both individual and between litters, of the embryos to parenterally administered agents. It is suggested that the blastocyst flat mount preparation could be adapted as a conveniently rapid and simple procedure for the screening of cytostatic and other growth-modifying agents.
Alkaline Phosphatase Activity in the Rat Uterus1
Authors: Prahlad K. V. , Conaway C. H.
Published: April 2021

A comparative study of the distribution of placental scars from the first and second pregnancies in laboratory rats indicated that the most posterior scar of first pregnancy tends to be nearer to the cervix than the most posterior second pregnancy scar (Momberg & Conaway, 1956). Frazer (1955) in an earlier study observed that more embryos implanted in the caudal half of the uterus of the rat than in the cranial half when the embryo number was four or less. Momberg & Conaway (1956) found this difference to be greater when the embryo number was high.
The length of S-phase and G2-phase of epithelial cells is regulated during growth and morphogenesis in Hydra attenuata
Authors: Herrmann Klaus , Berking Stefan
Published: April 2021

Feeding and sectioning of the freshwater polyp hydra generates signals that (1) shorten the G2-phase of epithelial cells and (2) prevent epithelial cells from entering mitosis for a short period of time. The latter effect appears to be mediated by inhibitor I, an endogenous morphogenetically active compound of hydra tissue. At least after feeding, the S-phase is shortened as well.
A probabilistic model of mosaicism based on the histological analysis of chimaeric rat liver
Authors: Iannaccone P. M. , Weinberg W. C. , Berkwits L.
Published: April 2021

The analysis of pattern development in mosaic and chimaeric animals has provided insight into a number of developmental problems. In order to aid the understanding of the dynamics of the development of mosaic tissues, a computer simulation of the generation of a mosaic tissue was created using simple probabilistic decisions. Results of quantitative analysis of the simulated mosaicism were compared with chimaeric liver. Chimaeric animals were produced by morula aggregation between histologically distinguishable strains of congenic rats. The livers of these animals revealed a pattern of patchy mosaicism unrelated to either acinar or lobular architecture of the organ. Independent quantifiable parameters were correlated and compared between the simulation and chimaeric liver tissue. This analysis showed that extensive cell migration is not required to develop finely variegated mosaic tissue and that the patterns of mosaicism observed could have resulted from tissue development in which as few as three reiterated decisions were required. First, the simulation established anlagen of two cell types of various specified proportions with randomly chosen placement. Second, in each generation of the simulation the order in which the cells divided was established randomly. Third, there was a random choice of the direction of placement of the daughter cell. The quantitative relationships between the proportion of cell types, the area of patches and the number of patches per unit area was consistent between the simulation and the chimaeric tissue.
The midblastula cell cycle transition and the character of mesoderm in u.v.-induced nonaxialXenopusdevelopment
Authors: Cooke Jonathan , Smith J. C.
Published: April 2021

Xenopus embryos (UV embryos) resulting from u.v. (254 nm) irradiation to the vegetal egg hemisphere and thus developing little or no axial pattern (UV5 -Scharf & Gerhart, 1983), have been compared histologically with synchronous normal siblings at each of three stages. In addition, the relative amounts of bloodforming tissue produced in normal and in UV embryos have been studied by Western blotting total protein from larval stages and by immunofluorescence on sections. The observations on midblastulae (around 5000 cells) were aimed at detecting any systematic retardation, due to u.v., of the slowing of the cell cycle that normally commences at the 2-4000 cell stage and makes possible zygotic transcription and the preparation for gastrulation. No such retardation was apparent. Observations on postgastrular stages gave an assessment of the size and character of the population of mesoderm founder cells, in relation to the control, for embryos visibly undergoing entirely nonaxial development. Little deficit in total mesodermal cell number was found, though the entire mesoderm adopted the histological character proper to only some 40 % of that in the normal pattern i.e. trunk lateral plate. Blood-forming capacity appears to be enhanced out of all proportion to the size of the mesoderm as a whole. The results are discussed in terms of the probable nature of the primary positional system for axial pattern and the later mechanisms of mesodermal patterning.
Interference in vimentin assembly in vitro by synthetic peptides derived from the vimentin head domain
Authors: Hofmann Ilse , Herrmann Harald
Published: April 2021

The importance of the amino-terminal domain (“head”) of type HI intermediate filament (IF) proteins in IF assembly has been examined by testing the influence of synthetic peptides representing a highly conserved decameric motif, , located near the amino terminus of vimentin. When added to soluble vimentin subunits this peptide induces, at fourfold molar excess or slightly above, the appearance of short, regular rod-like structures as determined by electron microscopy of negatively stained and rotary-shadowed preparations as well as by viscometry. At higher peptide concentrations large, irregularly shaped aggregates of mostly non-IF structures formed, but this aggregation was reversible by prolonged dialysis against low ionic strength buffer. The aggregating effect of this peptide was highly sequence-specific and was not seen with point-mutated sequences such as RR → TR or with unrelated peptides containing a central diarginine, indicating that it is not simply ionic. When different hexapeptides representing different “head” positions were compared, only the central sequence, SYRRXF, was as effective as the decamer.

The addition of peptide during IF assembly did not prevent filament formation, although 50-fold molar excess of peptide resulted in a drastic increase (up to 40 nm) in the width of the filaments, which also appeared less regular, thus reflecting some interference with assembly. In contrast to the effects on soluble vimentin, the decameric peptide did not disturb Ils, indicating that the binding domain is “masked” or stabilized in the filaments.

To identify the domain to which the peptide binds, three different binding assays using vimentin fragments and genetically engineered vimentin deletion mutants were employed. The results indicate that the binding domain of the near-amino-terminal peptide is located at the start of the α-helical “rod” domain of the protein. Possible mechanisms of interaction of these two portions of vimentin during IF assembly are discussed.
WASP and WAVE family proteins: key molecules for rapid rearrangement of cortical actin filaments and cell mov
Authors: Takenawa Tadaomi , Miki Hiroaki
Published: April 2021

Reorganization of cortical actin filaments plays critical roles in cell movement and pattern formation. Recently, the WASP and WAVE family proteins WASP and N-WASP, and WAVE1, WAVE2 and WAVE3 have been shown to regulate cortical actin filament reorganization in response to extracellular stimuli. These proteins each have a verprolin-homology (V) domain, cofilin-homology (C) domain and an acidic (A) region at the C-terminus, through which they activate the Arp2/3 complex, leading to rapid actin polymerization. N-WASP is usually present as an inactive form in which the VCA region is masked. Cooperative binding of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) exposes the VCA region, activating N-WASP. In addition to this activation mechanism, WISH also activates N-WASP independently of Cdc42 and PtdIns(4,5)P2, by binding to the proline-rich region of N-WASP. N-WASP activation induces formation of filopodia in vivo. In contrast, the ubiquitously expressed form of WAVE2 is activated downstream of Rac, leading to formation of lamellipodia. In this case, IRSp53 transmits a signal from Rac to WAVE2 through formation of a ternary Rac-IRSp53-WAVE2 complex. Thus, N-WASP, which is activated downstream of Cdc42 or independently by WISH, induces formation of filopodia and WAVE2, which is activated via IRSp53 downstream of Rac, induces formation of lamellipodia.
Integrins and cell proliferation
Authors: Schwartz Martin Alexander , Assoian Richard K.
Published: April 2021

Cell cycle progression in mammalian cells is strictly regulated by both integrin-mediated adhesion to the extracellular matrix and by binding of growth factors to their receptors. This regulation is mediated by G1 phase cyclin-dependent kinases (CDKs), which are downstream of signaling pathways under the integrated control of both integrins and growth factor receptors. Recent advances demonstrate a surprisingly diverse array of integrin-dependent signals that are channeled into the regulation of the G1 phase CDKs. Regulation of cyclin D1 by the ERK pathway may provide a paradigm for understanding how cell adhesion can determine cell cycle progression.
Specific domains drive VM32E protein distribution and integration inDrosophilaeggshell layers
Authors: Andrenacci Davide , Cernilogar Filippo M. , Taddei Carlo , Rotoli Deborah , Cavaliere Valeria , Graziani Franco , Gargiulo Giuseppe
Published: April 2021

A study was made of the localization and assembly of the VM32E protein, a putative vitelline membrane component of the Drosophila eggshell. The results highlight some unique features of this protein compared with the other proteins of the same gene family. At the time of its synthesis (stage 10), the VM32E protein is not detectable in polar follicle cells. However, it is able to move in the extracellular space around the oocyte and, by stage 11 is uniformly distributed in the vitelline membrane. During the terminal stages of oogenesis the VM32E protein is partially released from the vitelline membrane and becomes localized in the endochorion layer also. By analyzing transgenic flies carrying variously truncated VM32E proteins, we could identify the protein domains required for the proper assembly of the VM32E protein in the eggshell. The highly conserved vitelline membrane domain is implicated in the early interactions with other components and is required for cross-linking VM32E protein in the vitelline membrane. The terminal carboxylic domain is necessary for localization to the endochorion layer. Protein with the C-end domain deleted is localized solely to the vitelline membrane and cross-linked only in laid eggs, as occurs for the other vitelline membrane proteins.
Live observation of fission yeast meiosis in recombination-deficient mutants
Authors: Molnar Monika , Bähler Jürg , Kohli Jürg , Hiraoka Yasushi
Published: April 2021

Regular segregation of homologous chromosomes during meiotic divisions is essential for the generation of viable progeny. In recombination-proficient organisms, chromosome disjunction at meiosis I generally occurs by chiasma formation between the homologs (chiasmate meiosis). We have studied meiotic stages in living rec8 and rec7 mutant cells of fission yeast, with special attention to prophase and the first meiotic division. Both rec8 and rec7 are early recombination mutants, and in rec7 mutants, chromosome segregation at meiosis I occurs without any recombination (achiasmate meiosis). Both mutants showed distinct irregularities in nuclear prophase movements. Additionally, rec7 showed an extended first division of variable length and with single chromosomes changing back and forth between the cell poles. Two other early recombination deficient mutants (rec14 and rec15) showed very similar phenotypes to rec7 during the first meiotic division, and the fidelity of achiasmate chromosome segregation slightly exceeded the expected random level. We discuss possible regulatory mechanisms of fission yeast to deal with achiasmate chromosome segregation.
The interface of receptor trafficking and signalling
Authors: Clague Michael J. , Urbé Sylvie
Published: April 2021

The intimate relationship between receptor trafficking and signalling is beginning to reveal its secrets. Receptor endocytosis provides a mechanism for attenuation of signalling by transfer of receptors to degradative compartments. However, it can also determine signalling output by providing a different combination of downstream effectors at endocytic compartments compared with the plasma membrane. Rab5, Hrs and Cbl, are three examples of proteins that can influence both tyrosine kinase receptor trafficking and signalling pathways. By operating at this intersection, they are well placed to couple these aspects of cell function. Each element of the Rab5 GTPase cycle is influenced by signal transduction events, which will correspondingly influence recruitment of effector proteins and receptor distribution. Hrs and Cbl, which both undergo tyrosine phosphorylation in response to growth factor stimulation, are believed to influence receptor sorting in the early endosome and engage in multiple interactions, which may play a direct role in signalling cascades.
A specific targeting signal directs Runx2/Cbfa1 to subnuclear domains and contributes to transactivation of the osteocalcin gene
Authors: Zaidi S. Kaleem , Javed Amjad , Choi Je-Yong , van Wijnen André J. , Stein Janet L. , Lian Jane B. , Stein Gary S.
Published: April 2021

Key components of DNA replication and the basal transcriptional machinery as well as several tissue-specific transcription factors are compartmentalized in specialized nuclear domains. In the present study, we show that determinants of subnuclear targeting of the bone-related Runx2/Cbfa1 protein reside in the C-terminus. With a panel of C-terminal mutations, we further demonstrate that targeting of Runx2 to discrete subnuclear foci is mediated by a 38 amino acid sequence (aa 397-434). This nuclear matrix-targeting signal (NMTS) directs the heterologous Gal4 protein to nuclear-matrix-associated Runx2 foci and enhances transactivation of a luciferase gene controlled by Gal4 binding sites. Importantly, we show that targeting of Runx2 to the NM-associated foci contributes to transactivation of the osteoblast-specific osteocalcin gene in osseous cells. Taken together, these findings identify a critical component of the mechanisms mediating Runx2 targeting to subnuclear foci and provide functional linkage between subnuclear organization of Runx2 and bone-specific transcriptional control.
Control of the nuclear-cytoplasmic partitioning of annexin II by a nuclear export signal and by p11 binding
Authors: Eberhard David A. , Karns Larry R. , VandenBerg Scott R. , Creutz Carl E.
Published: April 2021

This study investigated mechanisms controlling the nuclear-cytoplasmic partitioning of annexin II (AnxII). AnxII and its ligand, p11, were localized by immunofluorescence to the cytoplasmic compartment of U1242MG cells, with minimal AnxII or p11 detected within nuclei. Similarly, GFP-AnxII and GFP-p11 chimeras localized to the endogenous proteins. Likewise, GFP-AnxII(1-22) was excluded from nuclei, whereas GFP-AnxII(23-338) and GFP alone were distributed throughout the cells. Immunoprecipitation and biochemical studies showed that GFP-AnxII did not form heteromeric complexes with endogenous p11 and AnxII. Thus, the AnxII N-tail is necessary and sufficient to cause nuclear exclusion of the GFP fusion protein but this does not involve p11 binding. A nuclear export signal consensus sequence was found in the AnxII 3-12 region. The consensus mutant GFP-AnxII(L10A/L12A) confirmed that these residues are necessary for nuclear exclusion. The nuclear exclusion of GFP-AnxII(1-22) was temperature-dependent and reversible, and the nuclear export inhibitor leptomycin B (LmB) caused GFP-AnxII or overexpressed AnxII monomer to accumulate in nuclei. Therefore, AnxII monomer can enter the nucleus and is actively exported. However, LmB had little effect on the localization of AnxII/p11 complex in U1242MG cells, indicating that the complex is sequestered in the cytoplasm. By contrast, LmB treatment of v-src-transformed fibroblasts caused endogenous AnxII to accumulate in nuclei. The LmB-induced nuclear accumulation of AnxII was accelerated by pervanadate and inhibited by genistein, suggesting that phosphorylation promotes nuclear entry of AnxII. Thus, nuclear exclusion of AnxII results from nuclear export of the monomer and sequestration of AnxII/p11 complex, and may be modulated by phosphorylation.
Cloning and expression of the putative aggregation factor from the marine spongeGeodia cydonium
Authors: Schütze Joachim , Krasko Anatoli , Diehl-Seifert Bärbel , Müller Werner E. G.
Published: April 2021

Sponges (phylum Porifera) have extensively been used as a model system to study cell-cell interaction on molecular level. Recently, we identified and cloned the putative aggregation receptor (AR) of the sponge Geodia cydonium, which interacts in a heterophilic way with the aggregation factor (AF) complex. In the present study, antibodies against this complex have been raised that abolish the adhesion function of the enriched sponge AF, the AF-Fraction 6B. Using this antibody as a tool, a complete 1.7 kb long cDNA, GEOCYAF, could be isolated from a cDNA library that encodes the putative AF. Its deduced aa sequence in the N-terminal section comprises high similarity to amphiphysin/BIN1 sequences found in Protostomia and Deuterostomia. However, the C-terminal portion of the sponge sequence lacks the SH3 domain characteristic for amphiphysin/BIN1. The polypeptide with a calculated size of 47 kDa was expressed in Escherichia coli. The recombinant, soluble 36 kDa putative AF was prepared and found to compete with the AF complex-associated adhesion protein of the AF-Fraction 6B for the binding sites at the cell surface. Furthermore, the recombinant putative AF was recognized by the antibody used to screen the cDNA library by western blotting. In addition, there is evidence that the recombinant putative AF binds to the G. cydonium galectin. It is concluded that the putative G. cydonium AF - a further autapomorphic molecule characteristic for Metazoa - binds to the AR present on the cell surface in association with the homologous galectin.
A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation
Authors: Munafó Daniela B. , Colombo María I.
Published: April 2021

Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic portions and intracellular organelles in a membrane vacuole called the autophagosome. These vesicles fuse with lysosomes and the sequestered material is degraded. Owing to the complexity of the autophagic pathway and to its inaccessibility to external probes, little is known about the molecular mechanisms that regulate autophagy in higher eukaryotic cells. We used the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker to analyze at the molecular level the machinery involved in the autophagic process. We have developed a morphological and biochemical assay to study authophagy in living cells based on the incorporation of MDC. With this assay we observed that the accumulation of MDC was specifically induced by amino acid deprivation and was inhibited by 3-methlyadenine, a classical inhibitor of the autophagic pathway. Additionally, wortmannin, an inhibitor of PI3-kinases that blocks autophagy at an early stage, inhibited the accumulation of MDC in autophagic vacuoles. We also found that treatment of the cells with N-ethylmaleimide (NEM), an agent known to inhibit several vesicular transport events, completely blocked the incorporation of MDC, suggesting that an NEM-sensitive protein is required for the formation of autophagic vacuoles. Conversely, vinblastine, a microtubule depolymerizing agent that induces the accumulation of autophagic vacuoles by preventing their degradation, increased the accumulation of MDC and altered the distribution and size of the autophagic vacuoles. Our results indicate that in the presence of vinblastine very large MDC-vacuoles accumulated mainly under starvation conditions, indicating that the expansion of autophagosomes is upregulated by amino acid deprivation. Furthermore, these MDC-vacuoles were labeled with LC3, one of the mammalian homologues of the yeast protein Apg8/Aut7 that plays an important role in autophagosome formation.
Characterization of functional domains of mDia1, a link between the small GTPase Rho and the actin cytoskeleton
Authors: Krebs Anja , Rothkegel Martin , Klar Martin , Jockusch Brigitte M.
Published: April 2021

The widely expressed diaphanous proteins, a subclass of formins, comprise links between the Rho GTPases and the actin-based cytoskeleton. They contain several functional domains that are thought to be responsible for interaction with different ligands: the FH1 domain for binding the actin-associated protein profilin; the RBD for targeting activated Rho; and the C-terminal CIID module for autoregulation of the overall diaphanous activity. Using deletion constructs of the murine mDia1, we have analyzed the functional properties of these three domains separately in in vitro assays and in transiently and stably transfected cell lines. We show that the proline-rich FH1 domain effectively binds to profilins in vitro as well as in cells, that the RBD complexes with the CIID in a species-restricted manner and that overexpression of RBD causes spontaneous ruffling and loss of stress fibers, together with loss of directional motility. Supertransfection of cells stably expressing the RBD with dominant negative Rac effectively suppresses ruffling. Our data contribute to the understanding of the function of these domains in linking the actin cytoskeleton with the Rho-signaling cascade. Furthermore, they suggest that inactivation of Rho by exogenous RBD causes upregulation of Rac activity in the transfected cells.