bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intermediate filament interactions can be altered by HSP27 and αB-crystallin
Authors: Perng Ming Der , Cairns Lindsay , van den Ijssel Paul , Prescott Alan , Hutcheson Aileen M. , Quinlan Roy A. 
Published: April 2021

ABSTRACT
HSP27 and αB-crystallin are both members of the small heat shock protein family. αB-crystalllin has been proposed to modulate intermediate filaments and recently a mutation in αB-crystallin has been identified as the genetic basis of desmin related myopathy. This disease is characterised in its pathology by aggregates of intermediate filaments associated with αB-crystallin. Here we report that HSP27 like αB-crystallin is associated with glial fibrillary acidic protein and vimentin intermediate filament networks in unstressed U373MG astrocytoma cells. HSP27 is also associated with keratin filaments in MCF7 cells, indicating that this association is not restricted to a particular intermediate filament type. The association of sHSPs with both the soluble and filamentous intermediate filament fractions of U373 cells was demonstrated biochemically. Heat shock or drug treatments induced a co-collapse of intermediate filaments and associated small heat shock proteins. These data show that the presence of HSP27 or αB-crystallin could not prevent filament collapse and suggest that the purpose of this association is more than just filament binding. Indeed, in U373MG cells the intermediate filament association with small heat shock proteins is similar to that observed for another protein chaperone, HSC70. In order to discern the effect of different chaperone classes on intermediate filament network formation and maintenance, several in vitro assays were assessed. Of these, falling ball viscometry revealed a specific activity of small heat shock proteins compared to HSC70 that was apparently inactive in this assay. Intermediate filaments form a gel in the absence of small heat shock proteins. In contrast, inclusion of αB-crystallin or HSP27 prevented gel formation but not filament assembly. The transient transfection of GFAP into MCF7 cells was used to show that the induction of a completely separate network of intermediate filaments resulted in the specific association of the endogenous HSP27 with these new GFAP filaments. These data lead us to propose that one of the major functions of the association of small heat shock proteins with intermediate filaments is to help manage the interactions that occur between filaments in their cellular networks. This is achieved by protecting filaments against those non-covalent interactions that result when they come into very close proximity as seen from the viscosity experiments and which have the potential to induce intermediate filament aggregation as seen in some disease pathologies.
Molecular architecture of the lens fiber cell basal membrane complex
Authors: Bassnett Steven , Missey Heather , Vucemilo Ivica 
Published: April 2021

ABSTRACT
Lens fiber cells are transparent, highly elongated, epithelial cells. Because of their unusual length these cells represent a novel model system to investigate aspects of epithelial cell polarity. In this study, we examined the fiber cell basal membrane complex (BMC). The BMC anchors fiber cells to the lens capsule and facilitates their migration across the capsule. Confocal microscopy revealed that bundled actin filaments converge beneath the center of each BMC and insert into the lateral membrane at points enriched in N-cadherin. Two other contractile proteins, caldesmon and myosin, were enriched in the BMC, co-localizing with f-actin bundles. The actin/N-cadherin complex formed a hexagonal lattice, cradling the posterior face of the lens. Removal of the capsule caused the tips of the fiber cells to break off, remaining attached to the stripped capsule. This provided a method for assaying cell adhesion and purifying BMC components. Fiber cell adhesion required Mg2+and/or Ca2+ and was disrupted by incubation with β1 integrin antibody. BMC proteins were compared with samples from the neighboring lateral membrane. Although some components were common to both samples, others were unique to the BMC. Furthermore, some lateral membrane proteins, most notably lens major intrinsic protein (MIP), were excluded from the BMC. Western blotting of BMC preparations identified several structural proteins originally found in focal adhesions and two kinases, FAK and MLCK, previously undescribed in the lens. These data suggest that the BMC constitutes a distinct membrane domain in the lens. The structural organization of the BMC suggests a role in shaping the posterior lens face and hence the refractive properties of the eye.
Sequential recruitment of NPC proteins to the nuclear periphery at the end of mitosis
Authors: Bodoor Khaldon , Shaikh Sarah , Salina Davide , Raharjo Wahyu Hendrati , Bastos Ricardo , Lohka Manfred , Burke Brian 
Published: April 2021

ABSTRACT
Nuclear pore complexes (NPCs) are extremely elaborate structures that mediate the bidirectional movement of macromolecules between the nucleus and cytoplasm. With a mass of about 125 MDa, NPCs are thought to be composed of 50 or more distinct protein subunits, each present in multiple copies. During mitosis in higher cells the nuclear envelope is disassembled and its components, including NPC subunits, are dispersed throughout the mitotic cytoplasm. At the end of mitosis, all of these components are reutilized. Using both conventional and digital confocal immunofluorescence microscopy we have been able to define a time course of post-mitotic assembly for a group of NPC components (CAN/Nup214, Nup153, POM121, p62 and Tpr) relative to the integral nuclear membrane protein LAP2 and the NPC membrane glycoprotein gp210. Nup153, a component of the nuclear basket, associates with chromatin towards the end of anaphase, in parallel with the inner nuclear membrane protein, LAP2. However, immunogold labeling suggests that the initial Nup153 chromatin association is membrane-independent. Assembly of the remaining proteins follows that of the nuclear membranes and occurs in the sequence POM121, p62, CAN/Nup214 and gp210/Tpr. Since p62 remains as a complex with three other NPC proteins (p58, 54, 45) during mitosis and CAN/Nup214 maintains a similar interaction with its partner, Nup84, the relative timing of assembly of these additional four proteins may also be inferred. These observations suggest that there is a sequential association of NPC proteins with chromosomes during nuclear envelope reformation and the recruitment of at least eight of these precedes that of gp210. These findings support a model in which it is POM121 rather than gp210 that defines initial membrane-associated NPC assembly intermediates.
Asymmetry of the spindle pole bodies and spg1p GAP segregation during mitosis in fission yeast
Authors: Cerutti Lorenzo , Simanis Viesturs 
Published: April 2021

ABSTRACT
In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is induced by a signal transduction network involving several protein kinases and a GTPase switch. One of the roles of the spg1p GTPase is to localise the cdc7p protein kinase to the poles of the mitotic spindle, from where the onset of septation is thought to be signalled at the end of mitosis. Immunofluorescence studies have shown that cdc7p is located on both spindle pole bodies early in mitosis, but only on one during the later stages of anaphase. This is mediated by inactivation of spg1p on one pole before the other. The GAP for spg1p is a complex of two proteins, cdc16p and byr4p. Localisation of cdc16p and byr4p by indirect immunofluorescence during the mitotic cell cycle showed that both proteins are present on the spindle pole body in interphase cells. During mitosis, byr4p is seen first on both poles of the spindle, then on only one. This occurs prior to cdc7p becoming asymmetric. In contrast, the signal due to cdc16p decreases to a low level during early mitosis, before being seen strongly on the same pole as byr4p. Double staining indicates that this is the opposite pole to that which retains cdc7p in late anaphase. Examination of the effect of inactivating cdc16p at various stages of the cell cycle suggests that cdc16p, together with cdc2p plays a role in restraining septum formation during interphase. The asymmetric inactivation of spg1p is mediated by recruitment of the cdc16p-byr4p GAP to one of the poles of the spindle before the other, and the asymmetry of the spindle pole bodies may be established early during mitosis. Moreover, the spindle pole bodies appear to be non-equivalent even after division has been completed.
The role of anillin in meiotic cytokinesis of Drosophila males
Authors: Giansanti Maria Grazia , Bonaccorsi Silvia , Gatti Maurizio 
Published: April 2021

ABSTRACT
Anillin is a 190 kDa actin-binding protein that concentrates in the leading edges of furrow canals during Drosophila cellularization and in the cleavage furrow of both somatic and meiotic cells. We analyzed anillin behavior during D. melanogaster spermatogenesis, and focused on the relationships between this protein and the F-actin enriched structures. In meiotic anaphases anillin concentrates in a narrow band around the cell equator. Cytological analysis of wild-type meiosis and examination of mutants defective in contractile ring assembly (chickadee and KLP3A), revealed that the formation of the anillin cortical band occurs before, and does not require the assembly of the F-actin based contractile ring. However, once the acto-myosin ring is assembled, the anillin band precisely colocalizes with this cytokinetic structure, accompanying its contraction throughout anaphase and telophase. In chickadee and KLP3A mutant ana-telophases the cortical anillin band fails to constrict, indicating that its contraction is normally driven by the cytokinetic ring. These findings, coupled with the analysis of anillin behavior in twinstar mutants, suggested a model on the role of anillin during cytokinesis. During anaphase anillin would concentrate in the cleavage furrow before the assembly of the contractile ring, binding the equatorial cortex, perhaps through its carboxy-terminal pleckstrin homology (PH) domain. Anillin would then interact with the actin filaments of the acto-myosin ring through its actin-binding domain, anchoring the contractile ring to the plasma membrane throughout cytokinesis.
Tau regulates the attachment/detachment but not the speed of motors in microtubule-dependent transport of single vesicles and organelles
Authors: Trinczek B. , Ebneth A. , Mandelkow E.-M. , Mandelkow E. 
Published: April 2021

ABSTRACT
We have performed a real time analysis of fluorescence-tagged vesicle and mitochondria movement in living CHO cells transfected with microtubule-associated protein tau or its microtubule-binding domain. Tau does not alter the speed of moving vesicles, but it affects the frequencies of attachment and detachment to the microtubule tracks. Thus, tau decreases the run lengths both for plus-end and minus-end directed motion to an equal extent. Reversals from minus-end to plus-end directed movement of single vesicles are strongly reduced by tau, but reversals in the opposite direction (plus to minus) are not. Analogous effects are observed with the transport of mitochondria and even with that of vimentin intermediate filaments. The net effect is a directional bias in the minus-end direction of microtubules which leads to the retraction of mitochondria or vimentin IFs towards the cell center. The data suggest that tau can control intracellular trafficking by affecting the attachment and detachment cycle of the motors, in particular by reducing the attachment of kinesin to microtubules, whereas the movement itself is unaffected.
Nuclear pore localization and nucleocytoplasmic transport of eIF-5A: evidence for direct interaction with the export receptor CRM1
Authors: Rosorius Olaf , Reichart Beate , Krätzer Friedrich , Heger Peter , Dabauvalle Marie-Christine , Hauber Joachim 
Published: April 2021

ABSTRACT
Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain the unusual amino acid hypusine. The exact in vivo function of eIF-5A, however, is to date unknown. The finding that eIF-5A is an essential cofactor of the human immunodeficiency virus type 1 (HIV-1) Rev RNA transport factor suggested that eIF-5A is part of a specific nuclear export pathway. In this study we used indirect immunofluorescence and immunogold electron microscopy to demonstrate that eIF-5A accumulates at nuclear pore-associated intranuclear filaments in mammalian cells and Xenopus oocytes. We are able to show that eIF-5A interacts with the general nuclear export receptor, CRM1. Furthermore, microinjection studies in somatic cells revealed that eIF-5A is transported from the nucleus to the cytoplasm, and that this nuclear export is blocked by leptomycin B. Our data demonstrate that eIF-5A is a nucleocytoplasmic shuttle protein.
Chmadrin: A novel Ki-67 antigen-related perichromosomal protein possibly implicated in higher order chromatin structure
Authors: Takagi Masatoshi , Matsuoka Yosuke , Kurihara Toshinao , Yoneda Yoshihiro 
Published: April 2021

ABSTRACT
A novel perichromosomal protein, which we have named chmadrin, was identified from rat kangaroo PtK2 cells. The deduced amino acid sequence revealed structural homologies in several limited regions to the Ki-67 antigen (pKi-67). The subcellular localization of chmadrin was found to be similar to that of pKi-67 throughout the cell cycle, that is, predominantly nucleolar during interphase and perichromosomal in the mitotic phase. In addition, a certain population of the protein was found to be localized in heterochromatic foci in interphase nuclei. Transient expression analysis of the truncated proteins corresponding to the conserved regions clearly demonstrated the structural basis for the characteristic cellular localization. Residues 494-778, which show extensive similarity to the corresponding region of pKi-67, were efficiently targeted to nucleoli, whereas a repetitive structure found at the C-terminal portion, whose similarity to pKi-67 is weak, was localized precisely to mitotic chromosomes. The C-terminal portion was designated the ‘LR domain’ since several LR (leucine and arginine) pairs commonly appear in chmadrin and pKi-67. When overproduced in the interphase nuclei, the LR domain induced the formation of aberrant heterochromatin as a structural constituent. These are the first empirical data suggesting the involvement of perichromosomal proteins in the organization of chromatin structure.
F-actin as a functional target for retro-retinoids: A potential role in anhydroretinol-triggered cell death
Authors: Korichneva Irina , Hämmerling Ulrich 
Published: April 2021

ABSTRACT
The retro-retinoids, metabolites of vitamin A (retinol), belong to a family of lipophilic signalling molecules implicated in regulation of cell growth and survival. Growth-promoting properties have been ascribed to 14-hydroxy-retro-retinol (14HRR), while anhydroretinol (AR) was discovered to act as a natural antagonist triggering growth arrest and death by apoptosis. Based on morphological studies and inhibition of apoptosis by the kinase blocker, herbimycin A, it has been suggested that retro-retinoids exhibit their function in the cytosolic compartment. F-actin emerged as a functional target for retro-retinoid action. By FACS analysis and fluorescence microscopy of phalloidin-FITC labeled cells we demonstrated that F-actin reorganization was an early event in AR-triggered apoptosis. Fluorescence images of AR-treated fibroblasts displayed short, thick, stick-like and
punctate structures, and membrane ruffles at the cell periphery along with an increased diffuse staining pattern. Reversal of the AR effect by 14HRR or retinol indicates that F-actin is a common site for regulation by retro-retinoids. Inhibition of both cell death and actin depolymerisation by bcl-2 implies that cytoskeleton reorganization is downstream of bcl-2-related processes. Furthermore, stabilization of microfilaments by jasplakinolide increased the survival potential of AR treated cells, while weakening the cytoskeleton by cytochalasin B abetted apoptosis. Thus the cytoskeleton is an important way station in a communication network that decides whether a cell should live or die.
The Emery-Dreifuss muscular dystrophy phenotype arises from aberrant targeting and binding of emerin at the inner nuclear membrane
Authors: Fairley Elizabeth A. L. , Kendrick-Jones John , Ellis Juliet A. 
Published: April 2021

ABSTRACT
The product of the X-linked Emery-Dreifuss muscular dystrophy gene is a single-membrane-spanning protein called emerin, which is localized to the inner nuclear membrane of all tissues studied. To examine whether a number of the mutant forms of emerin expressed in patients are mislocalized, we transfected GFP-emerin cDNA constructs reflecting these mutations into undifferentiated C2C12 myoblasts and showed that both wild type and all the mutant emerins are targeted to the nuclear membrane, but the mutants to a lesser extent. Mutant Del236-241 (deletion in transmembrane region) was mainly expressed as cytoplasmic aggregates, with only trace amounts at the nuclear envelope. Complete removal of the transmembrane region and C-terminal tail relocated emerin to the nucleoplasm. Mutations in emerin’s N-terminal domain had a less severe effect on disrupting nuclear envelope targeting. This data suggests that emerin contains multiple non-overlapping nuclear-membrane-targeting determinants.
Analysis of material immunoisolated using emerin antibodies, from either undifferentiated C2C12 myoblasts or purified hepatocyte nuclei, demonstrated that both A- and B-type lamins and nuclear actin interact with emerin. This is the first report of proteins interacting with emerin. The EDMD phenotype can thus arise by either the absence or a reduction in emerin at the nuclear envelope, and both of these disrupt its interactions with that of structural components of the nucleus. We propose that an emerin-nuclear protein complex exists at the nuclear envelope and that one of its primary roles is to stabilize the nuclear membrane against the mechanical stresses that are generated in muscle cells during contraction.
The α3 laminin subunit, α6β4 and α3β1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin
Authors: Goldfinger Lawrence E. , Hopkinson Susan B. , deHart Gregory W. , Collawn Sherry , Couchman John R. , Jones Jonathan C. R. 
Published: April 2021

ABSTRACT
Previously, we demonstrated that proteolytic processing within the globular domain of the α3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the α3 laminin subunit. This epitope is lost from the α3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix of cells that fail to process the α3 laminin subunit, but does not recognize the matrix of confluent cultures of MCF-10A cells, which efficiently process their α3 laminin chain. In subconfluent populations of MCF-10A cells, 12C4 only stains matrix deposited at the outer edges of cell colonies. In these cells, integrin α3β1 occasionally colocalizes with the staining generated by the 12C4 antibody but α6β4 integrin does not. In wounded MCF-10A cell cultures, the 12C4 antibody stains the extracellular matrix beneath those cells at the very edge of the cellular sheet that moves to cover the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed α3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using α3 laminin subunit and integrin function-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (α6β4 and α3β1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results.
The nuclear envelope serves as an intermediary between the ER and Golgi complex in the intracellular parasite Toxoplasma gondii
Authors: Hager Kristin M. , Striepen Boris , Tilney Lewis G. , Roos David S. 
Published: April 2021

ABSTRACT
Morphological examination of the highly polarized protozoan parasite Toxoplasma gondii suggests that secretory traffic in this organism progresses from the endoplasmic reticulum to the Golgi apparatus using the nuclear envelope as an intermediate compartment. While the endoplasmic reticulum is predominantly located near the basal end of the parasite, the Golgi is invariably adjacent to the apical end of the nucleus, and the space between the Golgi and nuclear envelope is filled with numerous coatomer-coated vesicles. Staining with antiserum raised against recombinant T. gondii β-COP confirms its association with the apical juxtanuclear region. Perturbation of protein secretion using brefeldin A, microtubule inhibitors or dithiothreitol disrupts the Golgi, causing swelling of the nuclear envelope, particularly at its basal end. Prolonged drug treatment leads to gross distention of the endoplasmic reticulum, filling the basal end of the parasite. Cloning and sequencing of the T. gondii homolog of the chaperonin protein BiP identifies the carboxy-terminal amino acid sequence HDEL as this organism’s endoplasmic reticulum-retention signal. Appending the HDEL motif to a recombinant secretory protein (a chimera between the parasite’s major surface protein fusion, P30, and the Green Fluorescent Protein) causes this secretory reporter to be retained intracellularly. P30-GFP-HDEL fluorescence was most intense within the nuclear envelope, particularly at the apical end. These data support a model of secretion in which protein traffic from the endoplasmic reticulum to Golgi occurs via the apical end of the nuclear envelope.
The casein kinase Iα isoform is both physically positioned and functionally competent to regulate multiple events of mRNA metabolism
Authors: Gross Stefan D. , Loijens Joost C. , Anderson Richard A. 
Published: April 2021

ABSTRACT
Casein kinase I is a highly conserved family of serine/threonine protein kinases present in every organism tested from yeast to humans. To date, little is known about the function of the higher eukaryotic isoforms in this family. The CKI isoforms in Saccharomyces cerevisiae, however, have been genetically linked to the regulation of DNA repair, cell cycle progression and cytokinesis. It has also been established that the nuclear localization of two of these isoforms is essential for their function. The work presented here demonstrates that the higher eukaryotic CKIα isoform is also present within nuclei of certain established cell lines and associated with discrete nuclear structures. The nature of its nuclear localization was characterized. In this regard, CKIα was shown to colocalize with factors involved in pre-mRNA splicing at nuclear speckles and that its association with these structures exhibited several biochemical properties in common with known splicing factors. The kinase was also shown to be associated with a complex that contained certain splicing factors. Finally, in vitro, CKIα was shown to be capable of phosphorylating particular splicing factors within a region rich in serine/arginine dipeptide repeat motifs suggesting that it has both the opportunity and the capacity to regulate one or more steps of mRNA metabolism.