bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cytoarchitecture of size-excluding compartments in living cells
Authors: Provance D. William , McDowall Alasdair , Marko Michael , Luby-Phelps Katherine 
Published: April 2021

ABSTRACT
By fluorescence ratio imaging of large and small inert tracer particles in living cells, we have previously shown that particles 24 nm in radius are excluded from other-wise uncharacterized compartments in the distal and perinuclear cytoplasm (Luby-Phelps, K. and Taylor, D.L., 1988. Cell Motil. Cytoskel. 10, 28-37). In this study we examined the cytoarchitecture of these compartments. Whole-mount TEM showed that distal size-excluding compartments were devoid of membrane-bounded organelles and were filled with a dense cytomatrix consisting of numerous, long bundles of thin filaments interconnected by a more random meshwork of short thin filaments. The mean diameter of void spaces in the cytomatrix of distal excluding compartments was 31 nm, compared to 53 nm in adjacent nonexcluding domains. The height of the distal excluding compartments was generally ≤ 50% of the height in the adjacent non-excluding compartment. An electrondense structure having the same projected outline as the perinuclear size-excluding compartment was visible by whole-mount TEM, but the cells were too thick and osmiophilic in this region to resolve any detail. Immunofluorescence localization of cytoskeletal proteins in distal excluding compartments indicated the presence of filament bundles containing F-actin, nonmuscle filamin (ABP280) and α-actinin. F-actin and ABP280, but not α-actinin, were found also in between these filament bundles. Microtubules and vimentin generally were rare or absent from distal excluding domains. Staining of living cells with DMB-ceramide revealed that the perinuclear size-excluding compartment consisted of a compact, juxtanuclear domain coinciding with the transGolgi, surrounded by a more diffuse domain coinciding with a perinuclear concentration of endoplasmic reticulum. Intense immunofluorescence staining for vimentin was also observed in the perinuclear size-excluding compartment. We propose that the most likely mechanism for exclusion from distal compartments is molecular sieving by a meshwork of actin filament bundles interconnected by an F-actin/ABP280 gel network, while exclusion from the perinuclear compartment may be due to close apposition of cisternae in the trans-Golgi and a network or basket of vimentin filaments in the centrosomal region of the cell.
Recycling of a secretory granule membrane protein after stimulated secretion
Authors: Hurtley Stella M. 
Published: April 2021

ABSTRACT
Recycling of a secretory granule membrane protein, dopamine-β-hydroxylase, was examined in primary cultures of bovine adrenal chromaffin cells. Cells were stimulated to secrete in the presence of antibodies directed against the luminal domain of dopamine-hydroxylase. The location of the antibodies after various times of reincubation and after a second secretory stimulus was assessed using immunofluorescence microscopy. Stimulation led to the exposure of dopamine-β-hydroxylase at the plasma membrane, which could be detected by a polyclonal antibody in living and fixed cells. The plasma membrane dopamine-β-hydroxylase, either alone or complexed with antibody, was rapidly internalized after removal of the secretagogue. Internalized protein-antibody complex remained stable for at least 24 hours of reculture. Twenty four hours after stimulation the cells with internalized anti-body could respond to further stimulation and some of the antibody was re-exposed at the plasma membrane. These findings were confirmed using FACS analysis. This suggests that the antibody-protein complex had returned to secretory granules that could respond to further secretagogue stimulation.
Keratocyte networks visualised in the living cornea using vital dyes
Authors: Poole C. Anthony , Brookes Nigel H. , Clover Gillian M. 
Published: April 2021

ABSTRACTFluorescent viability probes have been used to visualise and investigate the viability, morphology and organisation of the keratocyte within the stroma of the intact living cornea. The live cell probe, calcien-AM, in combination with a dead cell probe, ethidium homodimer (Live/Dead Assay, Molecular Probes, U.S.A.) proved superior to earlier generation vital dyes such as fluorescein diacetate or 5,6-carboxyfluorescein diacetate, initially used in combination with ethidium bromide. The ubiquitous distribution of esterase enzymes that cleave calcien-AM within the keratocyte cytoplasm produced a high concentration of fluorescently active calcein throughout the cell, including fine cell processes. Epi-illuminated fluorescence microscopy on transparent corneal dissections subsequently revealed details of keratocyte microanatomy and three-dimensional network organisation in situ. Three morphologically discrete subpopulations of keratocytes were identified: two formed relatively small bands of cells, immediately subjacent to either Bowman’s or Descemet’s membranes, the third subpopulation constituting the majority of keratocytes typically located within the corneal stroma. The results indicate that calcein-AM is able to penetrate intact living cornea revealing cell viability, and it also has the capacity to ‘trace’ cellular elements and reveal fine structure within a dense connective tissue matrix.
A centrosome-associated antibody from Drosophila melanogaster reveals a new microtubule-dependent structure in the equatorial zone of Parascaris univalens embryos
Authors: Jiménez M. , Goday C. 
Published: April 2021

ABSTRACT
The distribution of antigens to two antibodies (Bx63 and Rb188) that associate to Drosophila. melanogaster cen-trosomes has been investigated in the nematode Paras-caris. By western blot analysis both antibodies identify in Parascaris polypeptides of the same molecular mass as in Drosophila (Rb188 a 185 kDa antigen and Bx63 185 kDa and 66 kDa antigens). By immunocytochemistry we show that the centrosomes of Parascaris con-tain the 185 kDa antigen recognized by polyclonal Rb188 and monoclonal Bx63 antibodies. In addition, Bx63 reveals cytoplasmic midzone structures, not found in Drosophila, that display a cell cycle-dependent organization in embryos. These structures, which most prob-ably contain the 66 kDa antigen revealed by Bx63, appear at the onset of anaphase as fibrillar-like structures that during anaphase form a ring-like structure encircling the equatorial plane of the blastomere. Before furrowing, the antigen participates in the formation of the midbody and associates with convergent polar microtubules. After blastomere division, Bx63 signal persists as a single body between the daughter cells. The analysis of chilled and nocodazole-treated embryos suggests that the localization of the midzone Bx63 anti-gen is dependent on non-kinetochore microtubules. Inhibition of furrowing by cytochalasin B shows that the antigen persists after the disassembly of microfilaments. Cytological observations of contractile ring and Bx63 ring assembly indicate that both structures do not simul-taneously colocalize at the equatorial zone. The data suggest a spindle-dependent distribution of the Bx63 antigen during cytokinesis. We discuss the participation of this antigen in the organization of the midbody before furrowing, and consider the possible relevance of the midbody with respect to cell to cell communication during early development in nematodes.
Titin aggregates associated with intermediate filaments align along stress fiber-like structures during human skeletal muscle cell differentiation
Authors: Ven Peter F. M. van der , Schaart Gert , Croes Huib J. E. , Jap Paul H. K. , Ginsel Leo A. , Ramaekers Frans C. S. 
Published: April 2021

ABSTRACT
Differentiating human skeletal muscle cell cultures were used to study the association of titin with other sarcomeric and cytoskeletal proteins during myofibrillo-genesis. Several developmental stages of these cultures were double stained with antibodies to titin in combination with antibodies to α-actin, α-actinin, myosin heavy chain (MHC), nebulin, desmin, and β-tubulin. The first indications of titin expression were found in postmitotic mononuclear myoblasts where it is located in a random, punctate fashion. At the light microscope level no evidence was found for an association of these titin spots with any of the other proteins studied, with the exception of MHC, which colocalized with titin in a small minority of the titin expressing cells. Subsequently the titin spots were found to be linked to longitudinally oriented stress fiber-like structures (SFLS), containing-actinin and sarcomeric α-actin, but not MHC, nebulin or desmin. Upon further maturation titin antibodies seemed to stain SFLS in a rather homogeneous fashion together with MHC, α-actin and α-actinin. Thereafter a more periodic localization of titin, MHC, α-actin andα-actinin on SFLS became obvious. From these structures myofibrils developed as a result of further differentiation. Initially only short stretches with a striated titin, MHC, F-actin and α-actinin organization were found. Nebulin was integrated in these young myofibrils at a later developmental stage. Desmin was not found to be incorporated in these myofibrils until complete alignment of the sarcomeres in mature myotubes had occurred. At the ultrastructural level titin antibodies recognized aggregates that were associated with intermediate filaments (IF) in postmitotic mononuclear myoblasts. At a later maturational stage, prior to the development of cross-striated myofibrils, the IF-associated titin aggregates were found in close association with subsarcolemmally located SFLS. We conclude that IF and SFLS play an important role in the very early stages of in vitro human myofibrillogenesis. On the basis of our results we assume that titin aggregates are targeted to SFLS through IF. The association of titin with SFLS might be crucial for the unwinding of titin necessary for the assembly of sarcomeres and the first association of titin with other sarcomeric proteins.
Rab6 is associated with a compartment that transports rhodopsin from the trans-Golgi to the site of rod outer segment disk formation in frog retinal photoreceptors
Authors: Deretic Dusanka , Papermaster David S. 
Published: April 2021

ABSTRACT
The biogenesis of light sensitive membranes in retinal rod photoreceptors involves polarized sorting and tar-geting of newly synthesized rhodopsin to a specialized domain, the rod outer segment (ROS). We have isolated and characterized the population of post-Golgi mem-branes that mediate intracellular transport of rhodopsin. In the present study we have examined the association of small (20-25 kDa) GTP-binding (G) pro-teins with these membranes. We found that one of the small G proteins, rab6, behaves like an integral mem-brane protein of the post-Golgi vesicles, although ∼30% of rab6 is soluble. The distribution of the membrane-associated and the soluble forms is highly polarized. By confocal and EM immunocytochemistry it can be seen that most of rab6 is associated with the photoreceptor trans-Golgi cisternae, trans-Golgi network (TGN) and post-Golgi vesicles. The photoreceptor axon and synaptic terminal are unlabeled, but dendrites of deeper retinal layers are labeled. The distribution of rab6 across sucrose density gradient fractions parallels the distribution of sialyltransferase (a TGN marker) activity. About 9% of membrane-bound rab6 is associated, how-ever, with the rhodopsin-bearing sialyltransferase-free post-Golgi vesicles, which represent a very small fraction (&lt;1%) of the total retinal membranes. Rab6 is absent from the mature ROS disk membranes but it is present at the sites of new ROS disk formation and in the ROS cytoplasm. This suggests that rab6 becomes soluble upon disk membrane formation. Therefore, rab6 may function not only as a component of the sorting machinery of photoreceptors that delivers rhodopsin to its appropriate subcellular domain but may also par-ticipate in some aspects of ROS disk morphogenesis.
Diffusible, retinal factors stimulate the barrier properties of junctional complexes in the retinal pigment epithelium
Authors: Rizzolo Lawrence J. , Li Zhi-Qiang 
Published: April 2021

ABSTRACT
The retinal pigment epithelium lies at the interface between the neural retina and the choriocapillaris where it forms a blood-retinal barrier. Barrier function requires a polarized distribution of plasma membrane proteins and ‘tight’ tight junctions. During chicken embryogenesis, these features develop gradually. Although terminal junctional complexes are established by embryonic day 4, the distribution of the Na+/K+-ATPase is not polarized in all cells of the epithelium until embryonic day 11. Similarly, the tight junctions of early embryos are leaky, but become tight by hatching (embry-onic day 21). We used primary cell culture to examine the molecular basis of this gradual induction of polar-ized function. Pigment epithelium harvested from embryonic day 7, and cultured on filters, formed mono-layers coupled by junctional complexes. The distribution of the Na+/K+-ATPase was non-polarized and the tight junctions were leaky with a transepithelial electrical resistance of 20-30 cm2. To isolate diffusible factors that stimulate the transepithelial electrical resistance, neural retinas from embryonic day 7, 14 or 16 embryos were incubated at 37°C in base medium for 6 hours. The conditioned medium was added to the apical chamber of freshly cultured pigment epithelium. The distribution of the Na+/K+-ATPase became basolateral, and the electrical resistance gradually increased two to three times over 6 days. The increase in electrical resistance corresponded to a decrease in the rate of [3H]inulin diffusion across the monolayer. The effectiveness of the conditioned medium increased steadily with increasing age of the neural retina. Rather than increased production of an active factor, apparently different active factors were produced at different ages. The embryonic day 14 retinal-derived factors were heat-and alkali-labile and smaller than 10 kDa. Additionally, they were acid-stable and insensitive to proteolysis by cathepsin D. These studies provide evidence that diffusible factors from the neural retina regulate the gradual development of bar-rier function in the retinal pigment epithelium.
Transient effect of epidermal growth factor on the motility of an immortalized mammary epithelial cell line
Authors: Matthay Michael A. , Thiery Jean-Paul , Lafont Frank , Stampfer Martha F. , Boyer Brigitte 
Published: April 2021

ABSTRACT
The effects of growth factors on epithelial cell motility and dispersion have been examined on an immortalized human mammary epithelial cell line, the 184A1 non-tumorigenic cell line. Among all the molecules tested, epidermal growth factor (EGF) and transforming growth factor-α (TGF-α)were demonstrated to stimulate an increase in mammary epithelial cell motility and wound closure that was associated with a morphological transformation of the cells and was accompanied by modifications in cell-cell and cell-substrate adhesion systems. The EGF-induced increase in cell motility and monolayer wound closure occurred over a 24 hour period and was not dependent on an increase in cell number. The effect of EGF was abolished by inhibiting2 integrins with specific antibodies, indicating that part of the mechanism for the increase in cell motility and accelerated wound closure depends on α2 integrin functional expression. After 72 hours of exposure to EGF, the EGF-induced alterations in cell morphology, motility and cell adhesion systems underwent a spontaneous reversion that was correlated with a 10-fold reduction in the number of EGF receptors. The ability to regulate the scattering response induced by growth factors might be an important feature distinguishing normal epithelial cells from their tumoral counterparts.
Novel developmentally regulated exon identified in the rat fast skeletal muscle troponin T gene
Authors: Morgan Matthew J. , Earnshaw John C. , Dhoot Gurtej K. 
Published: April 2021

ABSTRACT
In theory, the rat fast skeletal muscle troponin T gene can generate 64 different isoforms. Here we report the identification of a novel alternative exon (exon y) that increases the potential isoform variation to 128. The inclusion of exon y in fast skeletal muscle troponin T mRNA occurs in perinatal, but not adult, skeletal muscle. Exon y is located between exons 8 and 9. This is the first time that a developmentally regulated exon located amongst a set of alternatively spliced exons has been described. Exon y is included in two mRNA iso-forms. The proteins that these mRNAs would encode have molecular masses greater than that of the largest fast skeletal muscle troponin T isoform lacking exon y. These two proteins correlate well in both size and pat-tern of expression with the two fast skeletal muscle troponin T isoforms expressed in perinatal skeletal muscle. These results indicate that there is coordinated regulation of the splicing of exon y with other alternative exons.
Repeated UVB irradiations do not have the same potential to promote stimulation of melanogenesis in cultured normal human melanocytes
Authors: Aberdam Edith , Roméro Christine , Ortonne Jean-Paul 
Published: April 2021

ABSTRACT
The major stimulus for human melanin production is ultraviolet (UV) radiation. Little is known about the mechanisms underlying this response and the eventual enzyme regulation resulting from this activation. We treated normal human melanocytes in culture with daily UVB radiations. Cumulative increases in UVB doses resulted in proportional increases in tyrosinase activity over the first few days whereas an intermittent pattern of tyrosinase activation was observed after the fifth day of irradiation. This intermittent pattern consisted of latency periods where no melanogenic response was elicited despite exposure to UVB. Tyrosinase activity in cellular extracts increased shortly after an effective irra-diation, peaked at 3 hours and thereafter decreased to below basal levels. Increased tyrosinase activity was associated with increased amounts of both the newly synthesized and mature forms of the enzyme. Decreased tyrosinase activity following an activation period was correlated with decreases in both the expression of tyrosinase mRNA and the amount of the newly synthe-sized form of the enzyme present in the melanocytes 24 hours after six irradiations. This particular pattern of stimulation of tyrosinase was not observed in S-91 murine melanoma cells after repeated UVB irradiations. Taken together these results may suggest a photo-pro-tective mechanism developed by irradiated normal human melanocytes.
The Cdc25 homologue Twine is required for only some aspects of the entry into meiosis in Drosophila
Authors: White-Cooper Helen , Alphey Luke , Glover David M. 
Published: April 2021

ABSTRACT
The twineHB5 mutation prevents spindle formation during the entry into meiosis in Drosophila males, but chromosome condensation and nuclear envelope break-down both still occur. This suggests the possibility that this particular cdc25 homologue is required to activate a p34cdc2 kinase required for only some of the events of this G2-M transition. In contrast, meiotic spindles do form in twineHB5 females, although these appear abnor-mal. However, the female meiotic divisions do not arrest at metaphase I as in wild type, but continue repeatedly, leading to gross non-disjunction. Small chromatin masses, corresponding in size to the fourth chromo-somes, often segregate properly to the spindle poles. These can persist into the embryos derived from twineHB5 females, where they appear to participate in mitotic divisions on thin spindles. In addition, these embryos contain a small number of large chromatin masses that are not associated with spindles.
Rous sarcoma virus-transformed cells develop peculiar adhesive structures along the cell periphery
Authors: Nakamura Norimasa , Tanaka Junya , Sobue Kenji 
Published: April 2021

ABSTRACT
Alteration of the cell/substratum adhesive structures of rat fibroblasts (3Y1 cells) upon transformation by Rous sarcoma virus (RSV) was investigated by immunofluo-rescence microscopy. In serum-containing culture medium, 3Y1 cells developed focal adhesions as their main adhesive structures, while BY1 cells expressed peculiar close contacts along the cell periphery with the vitronectin receptor integrin, in addition to podosomes. These peripheral close contacts are referred to as the peripheral adhesions. The peripheral adhesions were observed as a darker region than podosomes by inter-ference reflection microscopy. They were more easily destroyed by incubating the cells with RGD-containing peptide than were the focal adhesions. In contrast to focal adhesions and podosomes, actin bundles were not detected within the peripheral adhesions, where pp60v-src and tyrosine-phosphorylated proteins accumu-lated. Expression of the integrin was determined by the substratum composition when BY1 cells were cultured in serum-free culture medium. Under such conditions, BY1 cells expressed the peripheral adhesions within 3 hours on adhesion molecule-coated glass. On the other hand, in serum-containing medium, they first developed focal adhesions transiently at their early stage of adhe-sion, and then the peripheral adhesions were predomi-nantly expressed within 12 hours. Podosomes were formed in a time course similar to that of the periph-eral adhesions. These findings suggest that the periph-eral adhesion is a class of stable adhesive structure dis-tinct from the focal adhesion or podosome of BY1 cells. Similar close contact-type peripheral adhesions with the integrin were also observed in a variety of cultured cells such as normal fibroblasts at their logarithmic growth phase, phorbol ester-treated fibroblasts, and several malignant tumor cells, with poorly organized focal adhe-sions and stress fibers. These findings further suggest that the peripheral adhesions may be widely involved in the adhesion of cells that inadequately develop stress fibers and focal adhesions.
(Pro)collagenase (matrix metalloproteinase-1) is present in rodent osteoclasts and in the underlying bone-resorbing compartment
Authors: Delaissé J. M. , Eeckhout Y. , Neff L. , François-Gillet Ch. , Henriet P. , Su Y. , Vaes G. , Baron R. 
Published: April 2021

ABSTRACT
Osteoclasts resorb the extracellular matrix of bone by secreting enzymes and acid into a sealed-off compart-ment that they form upon attachment to the bone sur-face. Although the lysosomal cysteine proteinases can degrade collagen after the demineralization of bone at low pH, several lines of evidence suggest that collage-nase (matrix metalloproteinase-1, EC 3.4.24.7) may also be involved in this process. The question of whether col-lagenase is present in the osteoclast and/or in the bone-resorbing compartment has however not been resolved. We have prepared an antimouse collagenase antiserum and affinity-purified an IgG fraction that specifically immunoblots and immunoprecipitates (pro)collagenase. Using these antibodies, we demonstrate by immunolo-calization the presence of (pro)collagenase both in the osteoclasts and in the extracellular subosteoclastic bone-resorbing compartment. These specific localizations were observed not only in mice but also in rat and rabbit osteoclasts and using not only the antibody we have pre-pared but also antibodies raised in other laboratories against rat (Jeffrey et al., J. Cell. Physiol. 143, 396-403, 1990) and rabbit (Brinckerhoff et al., J. Biol. Chem. 265, 22262-22269, 1990) collagenase. Intracellular collage-nase was observed in the osteoclasts whether the cells were plated on bone or cultured on glass coverslips. It is proposed that osteoclastic collagenase is secreted in the resorbing compartment where it may cooperate with the lysosomal cysteine proteinases in the degradation of the collagen component of the matrix during the resorp-tion of bone.