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Cellular and Molecular Bioengineering Jun 2016Mechanical forces generated by nuclear-cytoskeletal coupling through the LINC (linker of nucleoskeleton and cytoskeleton) complex, an evolutionarily conserved molecular...
Mechanical forces generated by nuclear-cytoskeletal coupling through the LINC (linker of nucleoskeleton and cytoskeleton) complex, an evolutionarily conserved molecular bridge in the nuclear envelope (NE), are critical for the execution of wholesale nuclear positioning events in migrating and dividing cells, chromosome dynamics during meiosis, and mechanotransduction. LINC complexes consist of outer (KASH (, and homology)) and inner (SUN ()) nuclear membrane proteins. KASH proteins interact with the cytoskeleton in the cytoplasm and SUN proteins in the perinuclear space of the NE. In the nucleoplasm, SUN proteins interact with A-type nuclear lamins and chromatin-binding proteins. Recent structural insights into the KASH-SUN interaction have generated several questions regarding how LINC complex assembly and function might be regulated within the perinuclear space. Here we discuss potential LINC regulatory mechanisms and focus on the potential role of AAA+ (ATPases associated with various cellular activities) protein, torsinA, as a LINC complex regulator within the NE. We also examine how defects in LINC complex regulation by torsinA may contribute to the pathogenesis of the human neurological movement disorder, DYT1 dystonia.
PubMed: 27499815
DOI: 10.1007/s12195-016-0432-0 -
Histochemistry and Cell Biology Nov 2019Tuftelin was originally discovered and mostly studied in the tooth, but later found also in other organs. Despite its wide distribution among tissues, tuftelin's...
Tuftelin was originally discovered and mostly studied in the tooth, but later found also in other organs. Despite its wide distribution among tissues, tuftelin's function has so far been specified only in the formation of enamel crystals. Nevertheless, in many cases, tuftelin was suggested to be associated with cellular adaptation to hypoxia and recently even with cell differentiation. Therefore, we aimed to investigate tuftelin expression along with hypoxia-inducible factors (HIFs) during the early development of the mandibular/alveolar (m/a) bone, when osteoblasts started to differentiate in vivo and to compare their expression levels in undifferentiated versus differentiated osteoblastic cells in vitro. Immunohistochemistry demonstrated the presence of tuftelin already in osteoblastic precursors which were also HIF1-positive, but HIF2-negative. Nevertheless, HIF2 protein appeared when osteoblasts differentiated, one day later. This is in agreement with observations made with MC3T3-E1 cells, where there was no significant difference in tuftelin and Hif1 expression in undifferentiated vs. differentiated cells, although Hif2 increased upon differentiation induction. In differentiated osteoblasts of the m/a bone, all three proteins accumulated, first, prenatally, in the cytoplasm and later, particularly at postnatal stages, they displayed also peri/nuclear localization. Such a dynamic time-space pattern of tuftelin expression has recently been reported in neurons, which, as the m/a bone, differentiate under less hypoxic conditions as indicated also by a prevalent cytoplasmic expression of HIF1 in osteoblasts. However, unlike what was shown in cultured neurons, tuftelin does not seem to participate in final osteoblastic differentiation and its functions, thus, appears to be tissue specific.
Topics: 3T3 Cells; Animals; Cells, Cultured; Dental Enamel Proteins; Hypoxia-Inducible Factor 1; Immunohistochemistry; Mice; Osteogenesis; Transcription Factors
PubMed: 31520138
DOI: 10.1007/s00418-019-01813-4 -
Toxicology and Industrial Health Feb 2020In this study, the effects of a potent antioxidant, selenium, on apoptosis induced by acrolein, a cytotoxic and genotoxic environmental pollutant, were investigated by... (Comparative Study)
Comparative Study
In this study, the effects of a potent antioxidant, selenium, on apoptosis induced by acrolein, a cytotoxic and genotoxic environmental pollutant, were investigated by immunohistochemical and electron microscopic methods. One hundred adult male Wistar albino rats were used in the study. The rats were divided into four main groups: control, acrolein, selenium, and acrolein + selenium. The animals in the experimental groups were given 1 mg/kg/day selenium and 4 mg/kg/day acrolein daily for 7 days by gavage. After drug administration, each group was divided into subgroups according to the time they were to be euthanized: 12th hour, 1st, 2nd, 3rd, and 5th day. The rats in each group at the determined time were euthanized and their livers were removed. Routine histological procedures were performed for light and electron microscopy examinations. After applying the Terminal Deoxynucleotidyl Transferase dUTP nick end labeling assay on the liver sections, apoptotic index values were calculated. Comparing the liver sections of the rats in the acrolein group and the control group, acrolein was found to cause a significant increase in the apoptotic index. The apoptotic index values of the acrolein + selenium group decreased compared to the acrolein group. In the electron microscopic examinations, apoptotic findings were observed in the liver tissues of the rats given acrolein, such as chromatin condensation in the nucleus of hepatocytes, dilatations in the perinuclear space, and cytoplasmic vacuolization. These apoptotic findings were not observed in the acrolein + selenium group after the 12th hour. These findings show that selenium may potentially be useful as a protective agent for people exposed to acrolein.
Topics: Acrolein; Animals; Antioxidants; Apoptosis; Euthanasia, Animal; Liver; Male; Rats; Rats, Wistar; Selenium
PubMed: 32279646
DOI: 10.1177/0748233720909043 -
Atg39 links and deforms the outer and inner nuclear membranes in selective autophagy of the nucleus.The Journal of Cell Biology Feb 2022In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and...
In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to the forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane via its transmembrane domain and also associated with the inner nuclear membrane via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that autophagosome formation-coupled Atg39 crowding causes the NE to protrude toward the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 crowding in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.
Topics: Autophagy; Autophagy-Related Proteins; Chromosomes, Fungal; Nuclear Envelope; Receptors, Cytoplasmic and Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35061008
DOI: 10.1083/jcb.202103178 -
Pharmaceuticals (Basel, Switzerland) Jun 2022Linearolactone (LL) is a -clerodane type diterpene that has been shown to exert giardicidal effects; however, its mechanism of action is unknown. This work analyzes the...
Linearolactone (LL) is a -clerodane type diterpene that has been shown to exert giardicidal effects; however, its mechanism of action is unknown. This work analyzes the cytotoxic effect of LL on trophozoites and identifies proteins that could be targeted by this active natural product. Increasing concentrations of LL and albendazole (ABZ) were used as test and reference drugs, respectively. Cell cycle progression, determination of reactive oxygen species (ROS) and apoptosis/necrosis events were evaluated by flow cytometry (FCM). Ultrastructural alterations were analyzed by transmission electron microscopy (TEM). Ligand-protein docking analyses were carried out using the LL structure raised from a drug library and the crystal structure of an aldose reductase homologue (GdAldRed) from . LL induced partial arrest at the S phase of trophozoite cell cycle without evidence of ROS production. LL induced pronecrotic death in addition to inducing ultrastructural alterations as changes in vacuole abundances, appearance of perinuclear and periplasmic spaces, and deposition of glycogen granules. On the other hand, the in silico study predicted that GdAldRed is a likely target of LL because it showed a favored change in Gibbs free energy for this complex.
PubMed: 35890108
DOI: 10.3390/ph15070809 -
Mass Spectrometry (Tokyo, Japan) 2023Aberrant glycosylation of membrane proteins is a hallmark of cancer and a useful molecular marker for the diagnosis of breast cancer (BC). However, the molecular...
Aberrant glycosylation of membrane proteins is a hallmark of cancer and a useful molecular marker for the diagnosis of breast cancer (BC). However, the molecular mechanisms by which altered glycosylation affects the malignant transformations associated with BC are poorly understood. Accordingly, we performed comparative membrane -glycoproteomics using the human BC cell line pair, Hs578T, and its syngeneic normal cell line, Hs578Bst. A total of 359 -glycoforms derived from 113 proteins were identified in both cell lines, of which 27 were found only in Hs578T cells. Significant changes in -glycosylation were found in the lysosome-associated membrane protein 1 (LAMP1), the integrin family, and laminin. Confocal immunofluorescence microscopy images revealed the accumulation of lysosomes in the perinuclear space in cancer cells, which could be associated with marked changes in LAMP1 glycosylation, such as a decreased level of polylactosamine chains. Overall, the alterations in glycosylation may be involved in changes in the adhesion and degradation of BC cells.
PubMed: 37250596
DOI: 10.5702/massspectrometry.A0117 -
Cell Jun 2016Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a...
Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle "cloud" and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell's periphery. By drawing the endosomal system's architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.
Topics: Cell Line, Tumor; Dendritic Cells; Endoplasmic Reticulum; Endosomes; Humans; Intracellular Membranes; Macrophages; Neoplasm Proteins; Sequestosome-1 Protein; Transport Vesicles; Ubiquitin-Specific Proteases
PubMed: 27368102
DOI: 10.1016/j.cell.2016.05.078 -
Journal of Virology Sep 2023Nascent nucleocapsids of herpesviruses acquire a primary envelope during their nuclear export by budding through the inner nuclear membrane into the perinuclear space...
Nascent nucleocapsids of herpesviruses acquire a primary envelope during their nuclear export by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This process is mediated by a conserved viral heterodimeric complex designated the nuclear egress complex, which consists of the nuclear matrix protein and the nuclear membrane protein. In addition to its essential roles during nuclear egress, the nuclear matrix protein has been shown to interact with intracellular signaling pathway molecules including NF-κB and IFN-β to affect viral or cellular gene expression. The human herpesvirus 6A (HHV-6A) U37 gene encodes a nuclear matrix protein, the role of which has not been analyzed. Here, we show that HHV-6A U37 activates the heat shock element promoter and induces the accumulation of the molecular chaperone Hsp90. Mechanistically, HHV-6A U37 interacts with heat shock transcription factor 1 (HSF1) and induces its phosphorylation at Ser-326. We report that pharmacological inhibition of HSF1, Hsp70, or Hsp90 decreases viral protein accumulation and viral replication. Taken together, our results lead us to propose a model in which HHV-6A U37 activates the heat shock response to support viral gene expression and replication. IMPORTANCE Human herpesvirus 6A (HHV-6A) is a dsDNA virus belonging to the genus within the subfamily. It is frequently found in patients with neuroinflammatory disease, although its pathogenetic role, if any, awaits elucidation. The heat shock response is important for cell survival under stressful conditions that disrupt homeostasis. Our results indicate that HHV-6A U37 activates the heat shock element promoter and leads to the accumulation of heat shock proteins. Next, we show that the heat shock response is important for viral replication. Overall, our findings provide new insights into the function of HHV-6A U37 in host cell signaling and identify potential cellular targets involved in HHV-6A pathogenesis and replication.
Topics: Humans; Heat Shock Transcription Factors; Heat-Shock Response; Herpesvirus 6, Human; Viral Matrix Proteins; HSP90 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Promoter Regions, Genetic; Virus Replication; Phosphorylation; Gene Expression Regulation, Viral; Signal Transduction
PubMed: 37671864
DOI: 10.1128/jvi.00718-23 -
Cell Stress & Chaperones Jul 2019Skeletal myogenesis is a coordinated sequence of events associated with dramatic changes in cell morphology, motility, and metabolism, which causes cellular stress and...
Skeletal myogenesis is a coordinated sequence of events associated with dramatic changes in cell morphology, motility, and metabolism, which causes cellular stress and alters proteostasis. Chaperones, such as heat-shock proteins (HSPs), play important roles in limiting cellular stresses and maintaining proteostasis, but whether HSPs are specifically involved in myogenesis is not well understood. Here, we characterized gene and protein expression and subcellular localization of various HSPs in proliferating C2C12 myoblasts and differentiating myotubes under control conditions and in response to heat stress. Hsp25, Hsp40, and Hsp60 protein expression declined by 48, 35, and 83%, respectively, during differentiation. In contrast, Hsp70 protein levels doubled during early differentiation. Hsp25 was predominantly localized to the cytoplasm of myoblasts and myotubes but formed distinct aggregates in perinuclear spaces of myoblasts after heat-shock. Hsp40 was distributed diffusely throughout the cytoplasm and nucleus and, after heat-shock, translocated to the nucleus of myoblasts but formed aggregates in myotubes. Hsp60 localized to the perinuclear space in myoblasts but was distributed more diffusely across the cytoplasm in myotubes. Hsp70 was expressed diffusely throughout the cytoplasm and nucleus and translocated to the nucleus after heat-shock in myoblasts, but not in myotubes. Hsp90 was expressed diffusely across the cytoplasm in both myoblasts and myotubes under control conditions and did not change in response to heat-shock. These findings reveal distinct and different roles for HSPs in the regulation of myogenic cell proliferation and differentiation.
Topics: Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Heat-Shock Proteins; Heat-Shock Response; Muscle Development; Muscle Fibers, Skeletal; Myoblasts
PubMed: 31098840
DOI: 10.1007/s12192-019-01001-2 -
Biochemical and Biophysical Research... Jun 2020Apoptosis is the prototype for a regulated form of cell death, but recent studies have revealed other types of regulated forms of cell death, including necroptosis and...
Apoptosis is the prototype for a regulated form of cell death, but recent studies have revealed other types of regulated forms of cell death, including necroptosis and ferroptosis. The molecular mechanisms underlying the execution of these processes have been intensively investigated, yet the hallmarks of their morphology are not fully understood. Here, we report that electron lucent cytoplasm was a common feature of both necroptosis and ferroptosis, which was consistent with cytoplasmic vacuolization due to a defect in the cytoplasmic membrane integrity. Notably, the perinuclear space was dilated in necroptosis, but such dilation did not occur in ferroptosis. Cells undergoing ferroptosis, but not necroptosis, exhibited an electron lucent nucleus. We previously reported that one of the nuclear danger-associated molecular patterns (DAMPs), high mobility group box (HMGB)1, is rapidly released from the nucleus to the extracellular spaces of cells undergoing necroptosis through the ruptured nuclear and cytoplasmic membrane. Via time-lapse imaging of cells stably expressing HMGB1 fused to a fluorescence protein, we found that HMGB1 was also released from the nucleus to the cytosol, and then eventually released into the extracellular spaces in cells undergoing ferroptosis. Thus, nuclear membrane damage was induced prior to cytoplasmic membrane rupture in ferroptosis. Thus, dilation of the perinuclear space and an electron lucent nucleus may be the hallmarks of necroptosis and ferroptosis, respectively.
Topics: Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cytoplasm; Ferroptosis; HMGB1 Protein; Humans; Microscopy, Electron, Transmission; Necroptosis
PubMed: 32430176
DOI: 10.1016/j.bbrc.2020.04.127