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Reversible accumulation of endogenous tubulin in the nuclei of HeLa cells is inhibited by LMB. (A) Distribution of endogenous α/β-tubulin in HeLa cells under microtubule depolymerization conditions (nocodazole+cold) and after a temperature shift to normal temperature (temp. shift). (B) Effect of leptomycin B (LMB) on nuclear export of endogenous tubulin (for details see Materials and Methods). Cells were fixed and stained with anti-tubulin antibodies immediately after treatment as described in Materials and Methods. DNA was stained with Topro. Scale bars: 20 μm.
Source publication
We have investigated the subcellular distribution and dynamics of soluble tubulin in unperturbed and transfected HeLa cells. Under normal culture conditions, endogenous alpha/beta tubulin is confined to the cytoplasm. However, when the soluble pool of subunits is elevated by combined cold-nocodazole treatment and when constitutive nuclear export is...
Contexts in source publication
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... study, we compared the subcellular distribution of endogenous α/β-tubulin under normal conditions and under conditions that promote microtubule depolymerization, using immunofluorescence microscopy. When HeLa cells were incubated in the presence of nocodazole at 4 o C, α-and β-tubulin accumulated in the nucleus of most of the cells (82%) (see Fig. 1A, upper panels). However, nuclear tubulin exited the cell nucleus immediately after the cells were transferred to normal medium and cultured at 37 o C (Fig. 1A, lower panel), indicating nucleo-cytoplasmic shuttling that probably involves diffusion-driven import and active nuclear ...
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... using immunofluorescence microscopy. When HeLa cells were incubated in the presence of nocodazole at 4 o C, α-and β-tubulin accumulated in the nucleus of most of the cells (82%) (see Fig. 1A, upper panels). However, nuclear tubulin exited the cell nucleus immediately after the cells were transferred to normal medium and cultured at 37 o C (Fig. 1A, lower panel), indicating nucleo-cytoplasmic shuttling that probably involves diffusion-driven import and active nuclear ...
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... examine whether the nuclear export of endogenous soluble tubulin depends on previously identified export machinery (e.g. exportin 1), HeLa cells that had been pre-treated with nocodazole at 4 o C were transferred to 37 o C and cultured in the presence or absence of leptomycin B (LMB). As shown in Fig. 1B, no cells exhibiting nuclear staining could be identified in the control, but tubulin was retained in the nucleoplasm of LMB-treated cells. These results suggested an involvement of exportin 1 in the regulated export of soluble ...
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... overexpressing disNES-1 were clearly arrested in the G0-G1 phase, as indicated by the drop in G2-M figures and the significant increase of pre-S cells (Fig. 4D). Assessment of DAPI staining intensity in cells transfected with wild type or disNES-1 and in surrounding unperturbed cells showed no changes in chromatin state (supplementary material Fig. S1), indicating that G0-G1 arrest is not due to global chromatin condensation and a subsequent transcriptional block. Partitioning of cells in the different phases of the cell cycle did not change significantly after 24 hours (not shown), but the number of cells overexpressing disNES-1 dropped precipitously after 72 hours in culture (8% ...
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... association of tubulin with H3 in vivo was examined by immunoprecipitation. Using extracts from HeLa cells treated with nocodazole in the cold (to induce nuclear accumulation of tubulin as shown in Fig. 1A), we found that tubulin specifically co- precipitates with histone H3 (Fig. 5D). These results suggested that the interactions detected could be relevant to the nuclear localization and retention of soluble ...
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... cells grown on Petri dishes were incubated at 4 o C for 4 hours in the presence of nocodazole (33 μM for the first hour and 0.33 μM for the next 3 hours) to depolymerize microtubules and induce nuclear accumulation of α,β-tubulin (see Fig. 1). Cells were extracted with lysis buffer [50 mM HEPES (pH 7.5), 140 mM NaCl, 0.1% deoxycholate, 1% Triton X-100, 0.5 mM PMSF and protease inhibitors] for 10 minutes on ice. After sonication, the supernatant was clarified (12,000 g, 10 minutes, 4 o C), pre-absorbed on G-sepharose beads (GE Healthcare Bio-Sciences) and incubated with or ...
Citations
... Cell lysates from trophoblast cell lines and the resulting protein samples from different subcellular locations were collected using differential centrifugation and separated on SDS-PAGE before Western blotting analysis using markers for both the cytoplasmic and membrane components ( Figure 3A). Tubulin was used as a cytoplasmic marker [39] and was found to be significantly enriched in the total cell lysate samples compared to the membrane compartment for all samples tested ( Figure 3B), highlighting the fact there was little cytoplasmic contamination in the membrane fraction ( Figure 3A,B). Caveolin I, a well-established plasma membrane marker [40], on the other hand, was seen greatly enriched in the plasma membrane lane confirming the appropriate isolation of this cell compartment. ...
Whilst S100P has been shown to be a marker for carcinogenesis, we have shown, in non-physio-pathological states, that its expression promotes trophoblast motility and invasion but the mechanisms explaining these cellular processes are unknown. Here we identify the presence of S100P in the plasma membrane/cell surface of all trophoblast cells tested, whether lines, primary extravillous (EVT) cells, or section tissue samples using either biochemical purification of plasma membrane material, cell surface protein isolation through biotinylation, or microscopy analysis. Using extracellular loss of function studies, through addition of a specific S100P antibody, our work shows that inhibiting the cell surface/membrane-bound or extracellular S100P pools significantly reduces, but importantly only in part, both cell motility and cellular invasion in different trophoblastic cell lines, as well as primary EVTs. Interestingly, this loss in cellular motility/invasion did not result in changes to the overall actin organisation and focal adhesion complexes. These findings shed new light on at least two newly characterized pathways by which S100P promotes trophoblast cellular motility and invasion. One where cellular S100P levels involve the remodelling of focal adhesions whilst another, an extracellular pathway, appears to be focal adhesion independent. Both pathways could lead to the identification of novel targets that may explain why significant numbers of confirmed human pregnancies suffer complications through poor placental implantation.
... To test the impact of PARP3 activity on microtubule dynamics and markers of mitotic arrest in cells treated with eribulin, we assessed the effect of eribulin and olaparib alone or in combination on tubulin staining and nuclear morphology that have been associated with altered microtubule dynamics by eribulin and other microtubule targeting agents [35][36][37]41]. MDA-231 and MDA-436 24 hours after treatment with vehicle (CTL), eribulin IC 50 , 2 µM olaparib or the combination of eribulin plus olaparib. ...
Background:
Poly(ADP-ribose) polymerases 1 and 2 (PARP1, 2), and 3 mediate protein modifications that facilitate the recruitment of DNA repair factors to single and double strand breaks. PARP3 is unique in that it is also required for efficient mitotic progression and stabilization of the mitotic spindle. Eribulin, an anti-microtubule agent used clinically to treat breast cancer, exerts its cytotoxicity by altering microtubule dynamics resulting in cell cycle arrest and apoptosis. Herein, we hypothesize that the pan PARP inhibitor olaparib has the potential to enhance the cytotoxicity of eribulin by halting mitosis through inhibition of PARP3.
Methods:
The effect of olaparib on eribulin cytotoxicity was assessed using the Sulforhodamine (SRB) assay, with two triple negative breast cancer cell lines and an estrogen receptor positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer cell line. Alteration by the treatments on PARP3 activity and microtubule dynamics were assessed utilizing a chemiluminescent enzymatic assay and immunofluorescence, respectively. The effect of the treatments on cell cycle progression and apoptosis induction were assessed by flow cytometry using propidium iodide and Annexin V staining, respectively.
Results:
Our results demonstrate that non-cytotoxic concentrations of olaparib sensitize breast cancer cells regardless of ER status. Mechanistically, our results indicate that olaparib potentiates eribulin-induced cell cycle arrest at the G2/M boundary, PARP3 inhibition and microtubule destabilizing resulting in mitotic catastrophe and apoptosis.
Conclusions:
In breast cancer (regardless of ER status) settings, treatment outcomes could be improved by the incorporation of olaparib in eribulin treatment regimens.
... While the nuclear lamins are the main proteins of nucleus architecture and have a key role in maintaining the integrity of the nuclei, nuclear actin has been shown to be implicated in many of the processes that take place in the nucleus, including transcription, chromatin regulation, DNA damage repair, or long-range chromosome movement. The presence of another cytoskeleton component, αand β-tubulin, has been observed in the nuclei of some cancer cells or in other types of transformed cells, as well as in Xenopus oocytes [3][4][5]. However, the role of tubulin in the nucleoplasm has not been clarified as of yet. ...
The nuclear lamina is the main component of the nuclear cytoskeleton that maintains the integrity of the nucleus. However, it represents a natural barrier for viruses replicating in the cell nucleus. The lamina blocks viruses from being trafficked to the nucleus for replication, but it also impedes the nuclear egress of the progeny of viral particles. Thus, viruses have evolved mechanisms to overcome this obstacle. Large viruses induce the assembly of multiprotein complexes that are anchored to the inner nuclear membrane. Important components of these complexes are the viral and cellular kinases phosphorylating the lamina and promoting its disaggregation, therefore allowing virus egress. Small viruses also use cellular kinases to induce lamina phosphorylation and the subsequent disruption in order to facilitate the import of viral particles during the early stages of infection or during their nuclear egress. Another component of the nuclear cytoskeleton, nuclear actin, is exploited by viruses for the intranuclear movement of their particles from the replication sites to the nuclear periphery. This study focuses on exploitation of the nuclear cytoskeleton by viruses, although this is just the beginning for many viruses, and promises to reveal the mechanisms and dynamic of physiological and pathological processes in the nucleus.
... This reinforces the positive effect Noc had on XRCC1 recruitment. An earlier study (Akoumianaki et al., 2009) documented the trafficking of tubulin to the nucleus, its nuclear accumulation on Noc, and its ability to alter the interaction of histone H3 with other nuclear proteins. It was also reported that γ-tubulin interacts with Rad51 (Lesca et al., 2005), and that ATM, ATR, DNA-PK, MRN, p53, and 53BP1 all colocalize with cytoplasmic microtubules in the presence of vincristine, an inhibitor of microtubule turnover (Poruchynsky et al., 2015). ...
Upon induction of DNA damage with 405 nm laser light, proteins involved in Base Excision Repair (BER) are recruited to DNA lesions. We find that the dynamics of factors typical of either short-patch (XRCC1) or long-patch (PCNA) BER are altered by chemicals that perturb actin or tubulin polymerization in human cells. Whereas the destabilization of actin filaments by Latrunculin B, Cytochalasin B or Jasplakinolide decreases BER factor accumulation at laser-induced damage, inhibition of tubulin polymerization by Nocodazole increases it. We detect no recruitment of actin to sites of laser-induced DNA damage, yet the depolymerization of cytoplasmic actin filaments elevates both actin and tubulin signals in the nucleus. While published evidence suggested a positive role for F-actin in double-strand break repair in mammals, the enrichment of actin in budding yeast nuclei interferes with BER, augmenting sensitivity to Zeocin. Our quantitative imaging results suggest that the depolymerization of cytoplasmic actin may compromise BER efficiency in mammals not only due to elevated levels of nuclear actin, but also of tubulin. Our study is one of few linking cytoskeletal integrity to BER.
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... In the Cytoskeletal and structural category, several isoforms of keratin were identified distributed among all three samples, B, and C, in particular, 3 isoforms of K1(K1C10_RAT, K1C15_RAT, K1C19_RAT) and 7 isoforms of K2 (K22E_RAT, K2C1_RAT, K2C1B_RAT, K2C4_RAT, K2C5_RAT, K2C6A_RAT, K2C8_RAT) ( Figure 2); but only the actin ACTB_RAT isoform was identified as a peripheral nLD protein, interacting with the LD monolayer by electrostatic forces (Supplementary Table S4, Only-B fraction). In particular, cytoskeletal proteins such as actin and myosin have already been described and characterized in the cell nucleus in normal tissues and under physiological conditions [22,23], whereas keratins [24] and tubulin [25] have been identified in the nuclei of cancer and/or neoplastically transformed cells. ...
Nuclear-lipid droplets (nLD)-a dynamic cellular organelle that stores neutral lipids, within the nucleus of eukaryotic cells-consists of a hydrophobic triacylglycerol-cholesterol-ester core enriched in oleic acid (OA) surrounded by a monolayer of polar lipids, cholesterol, and proteins. nLD are probably involved in nuclear-lipid homeostasis serving as an endonuclear buffer that provides or incorporates lipids and proteins participating in signaling pathways, as transcription factors and enzymes of lipid metabolism and nuclear processes. In the present work, we analyzed the nLD proteome and hypothesized that nLD-monolayer proteins could be involved in processes similar as the ones occurring in the cLD including lipid metabolism and other cellular functions. We evaluated the rat-liver-nLD proteome under physiological and nonpathological conditions by GeLC-MS 2. Since isolated nLD are highly diluted, a protein-concentrating isolation protocol was designed. Thirty-five proteins were identified within the functional categories: cytoskeleton and structural, transcription and translation, histones, protein-folding and posttranslational modification, cellular proliferation and/or cancer, lipid metabolism, and transport. Purified nLD contained an enzyme from the lipid-metabolism pathway, carboxylesterase 1d (Ces1d/ Ces3). Nuclear Carboxylesterase localization was confirmed by Western blotting. By in-silico analyses rat Ces1d/ Ces3 secondary and tertiary structure predicted would be equivalent to human CES1. These results-the first nLD proteome-demonstrate that a tandem-GeLC-MS 2-analysis protocol facilitates studies like these on rat-liver nuclei. A diversity of cellular-protein function was identified indicating the direct or indirect nLD participation and involving Ces1d/Ces3 in the LD-population homeostasis.
... Before these findings, the generally accepted notion was that, with the exception of type V nuclear lamins, IF proteins strictly reside in the cytoplasm (Hobbs et al., 2016). Similar views were once held for other cytoskeletal proteins that have since been found to occur and function in the nucleus (Pereira et al., 1998;Yeh et al., 2004;Bettinger et al., 2004;Pederson, 2008;Akoumianaki et al., 2009;Castano et al., 2010). For example, actin adopts novel conformations in the nucleus, where it participates in chromatin remodeling, transcription, binding to nascent mRNA transcripts and mRNA export (Bettinger et al., 2004;Schoenenberger et al., 2005;Visa and Percipalle, 2010;Kapoor et al., 2013). ...
Keratin 17 ( KRT17 ; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal. Re-expression of wild-type K17, but not a mutant form lacking an intact nuclear localization signal (NLS), rescues nuclear morphology in KRT17 -null cells. Analyses of primary cultures of skin keratinocytes from a mouse strain expressing K17 with a mutated NLS corroborated these findings. Proteomics screens identified K17-interacting nuclear proteins with known roles in gene expression, chromatin organization and RNA processing. Key histone modifications and LAP2β (an isoform encoded by TMPO ) localization within the nucleus are altered in the absence of K17, correlating with decreased cell proliferation and suppression of GLI1 target genes. Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells.
This article has an associated First Person interview with the first author of the paper.
... Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells. Akoumianaki et al., 2009, Castano et al., 2010. For example, actin adopts novel conformations in the nucleus where it participates in chromatin remodeling, transcription via association with all three RNA polymerases, binding to nascent mRNA transcripts, and mRNA export (Bettinger et al., : bioRxiv preprint Figure S3. ...
Keratin 17 ( KRT17 ; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal. Re-expression of wildtype K17, but not a mutant form lacking an intact nuclear localization signal (NLS), rescues nuclear morphology in KRT17 null cells. Analyses of primary cultures of skin keratinocytes from a mouse strain expressing K17 with a mutated NLS corroborated these findings. Proteomics screens identified K17-interacting nuclear proteins with known roles in gene expression, chromatin organization, and RNA processing. Key histone modifications and LAP2β localization within the nucleus are altered in the absence of K17, correlating with decreased cell proliferation and suppression of GLI1 target genes. Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells.
Summary
Keratin 17 (K17) is one of two non-lamin intermediate filament proteins recently identified to localize to and function in the cell nucleus. K17 is here shown to regulate nuclear morphology, chromatin organization, LAP2 localization, and cell proliferation.
... Several cell-death-related processes, such as autophagy and apoptosis, which are activated under stress, rely on the microtubule system [71,72]. Still, the biological implications of the nuclear retention of MTOC-related proteins, which has been reported in cancer cells, have remained unclear [73,74]. ...
The interaction of oncogenes with cellular proteins is a major determinant of cellular transformation. The NUP98-HOXA9 and SET-NUP214 chimeras result from recurrent chromosomal translocations in acute leukemia. Functionally, the two fusion proteins inhibit nuclear export and interact with epigenetic regulators. The full interactome of NUP98-HOXA9 and SET-NUP214 is currently unknown. We used proximity-dependent biotin identification (BioID) to study the landscape of the NUP98-HOXA9 and SET-NUP214 environments. Our results suggest that both fusion proteins interact with major regulators of RNA processing, with translation-associated proteins, and that both chimeras perturb the transcriptional program of the tumor suppressor p53. Other cellular processes appear to be distinctively affected by the particular fusion protein. NUP98-HOXA9 likely perturbs Wnt, MAPK, and estrogen receptor (ER) signaling pathways, as well as the cytoskeleton, the latter likely due to its interaction with the nuclear export receptor CRM1. Conversely, mitochondrial proteins and metabolic regulators are significantly overrepresented in the SET-NUP214 proximal interactome. Our study provides new clues on the mechanistic actions of nucleoporin fusion proteins and might be of particular relevance in the search for new druggable targets for the treatment of nucleoporin-related leukemia.
... Actin, actin-binding proteins, including myosin isoforms as well as actin-related proteins (ARPs), are probably the best characterized in terms of their nuclear functions. However, evolving evidence that also components of the microtubules system are found in the cell nucleus (Akoumianaki et al., 2009) reflects an ever increasing complexity in the way cells work. In fact, seeing that components of microfilaments, microtubules and intermediate filaments are present in the cell nucleus will have to make us think differently about limiting the notion of a cytoskeleton only to the cytoplasm. ...
Cytoskeletal proteins are beginning to be considered as key regulators of nuclear function. Among them, actin and myosin have been implicated in numerous tasks, including chromatin regulation, transcription and assembly of nascent ribonucleoprotein complexes. We also know from work performed by several labs that influx of actin and myosin into the nucleus and out of the nucleus is tightly regulated. In particular, in the case of actin, its nucleocytoplasmic import/export cycle is controlled by the importin/exportin system and it correlates with the transcriptional state of the cell. These basic molecular functions of both actin and myosin seem to impact key cellular functions, including development and differentiation as well as the cellular response to DNA damage by directly affecting transcriptional reprograming. These observations are beginning to suggest that actin and myosin could play an important role in consolidating the organization of the mammalian genome and that loss of actin and myosin likely leads to a general instability of the genome. In this chapter, we provide a general background on evidence that actin and myosin are important in key nuclear functions. Following this, we will focus on evidence supporting of a role in genome organization and finally we will discuss increasingly striking results on the role of actin and myosin in the maintenance of genome integrity.
... Both actin and tubulin have been found to shuttle between cytoplasm and nucleus [57][58][59]. An emerging concept of 'nucleoskeleton' has been proposed to play a role in genome organization and gene regulation in cell differentiation and development [60]. ...
In the eukaryotic cell nucleus, cytoskeletal proteins are emerging as essential players in nuclear function. In particular, actin regulates chromatin as part of ATP-dependent chromatin remodeling complexes, it modulates transcription and it is incorporated into nascent ribonucleoprotein complexes, accompanying them from the site of transcription to polyribosomes. The nuclear actin pool is undistinguishable from the cytoplasmic one in terms of its ability to undergo polymerization and it has also been implicated in the dynamics of chromatin, regulating heterochromatin segregation at the nuclear lamina and maintaining heterochromatin levels in the nuclear interiors. One of the next frontiers is, therefore, to determine a possible involvement of nuclear actin in the functional architecture of the cell nucleus by regulating the hierarchical organization of chromatin and, thus, genome organization. Here, we discuss the repertoire of these potential actin functions and how they are likely to play a role in the context of cellular differentiation.
