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The linker of nucleoskeleton and cytoskeleton (LINC) complex has been implicated in various functions of the nuclear envelope, including nuclear migration, mechanotransduction and DNA repair. We previously revealed that the LINC complex component Sad1 and UNC84 domain containing 1 (SUN1) is required for sublethal-dose X-ray-enhanced cell migration and invasion. This study focused on epithelial-mesenchymal transition (EMT), which contributes to cell migration. Hence, the present study aimed to examine whether sublethal-dose X-irradiation induces EMT and whether LINC complex component SUN1 is involved in low-dose X-ray-induced EMT. This study showed that low-dose (0.5 Gy or 2 Gy) X-irradiation induced EMT in human breast cancer MDA-MB-231 cells. Additionally, X-irradiation increased the expression of SUN1. Therefore, SUN1 was depleted using siRNA. In SUN1-depleted cells, low-dose X-irradiation did not induce EMT. In addition, although the SUN1 splicing variant SUN1_916-depleted cells (containing 916 amino acids [AA] of SUN1) were induced EMT by low-dose X-irradiation like as non-transfected control cells, SUN1_888-depleted cells (which encodes 888 AA) were not induced EMT by low-dose X-irradiation. Moreover, since the Wnt/β-catenin signaling pathway regulates E-cadherin expression via the expression of the E-cadherin repressor Snail, the expression of β-catenin after X-irradiation was examined. After 24 hours of irradiation, β-catenin expression increased in non-transfected cells or SUN1_916-depleted cells, whereas β-catenin expression remained unchanged and did not increase in SUN1- or SUN1_888-depleted cells. Therefore, in this study, we found that low-dose X-irradiation induces EMT, and LINC complex component SUN1, especially SUN1_888, is required for X-ray-induced EMT via activation of the Wnt/β-catenin signaling pathway.

The evolutionarily conserved multiprotein complex THO/TREX is required for pre-mRNA processing, mRNA export and the maintenance of genome stability. In this study, we analyzed the genome-wide distribution of human THOC7, a component of human THO, by chromatin immunoprecipitation sequencing. The analysis revealed that human THOC7 occupies repetitive sequences, which include microsatellite repeats in genic and intergenic regions and telomeric repeats. The majority of the THOC7 ChIP peaks overlapped with those of the elongating form of RNA polymerase II and R-loops, indicating that THOC7 accumulates in transcriptionally active repeat regions. Knocking down THOC5, an RNA-binding component of human THO, by siRNA induced the accumulation of γH2AX in the repeat regions. We also observed an aberration in the telomeres in the THOC5-depleted condition. These results suggest that human THO restrains the transcription-associated instability of repeat regions in the human genome.

The nucleolar structure is highly dynamic and strictly regulated in response to internal cues, such as metabolic rates, and to external cues, such as mechanical forces applied to cells. Although the multilayered nucleolar structure is largely determined by the liquid-like properties of RNA and proteins, the mechanisms regulating the morphology and number of nucleoli remain elusive. The linker of the nucleoskeleton and cytoskeleton (LINC) complex comprises inner nuclear membrane Sad1/UNC-84 (SUN) proteins and outer nuclear membrane-localized nesprins. We previously showed that the depletion of SUN1 proteins affects nucleolar morphologies. This study focuses on the function of SUN1 splicing variants in determining nucleolar morphology. An RNA interference strategy showed that the predominantly expressed variants, SUN1_888 and SUN1_916, were crucial for nucleolar morphology but functionally distinct. In addition, the depletion of either SUN1_888 or SUN1_916 altered the chromatin structure and affected the distribution of histone modifications. Based on these results, we propose a model in which the LINC complex plays a role in modulating nucleolar morphology and numbers via chromatin.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is a multifunctional protein complex that is involved in various processes at the nuclear envelope, including nuclear migration, mechanotransduction, chromatin tethering and DNA damage response. We recently showed that a nuclear envelope protein, Sad1 and UNC84 domain protein 1 (SUN1), a component of the LINC complex, has a critical function in cell migration. Although ionizing radiation activates cell migration and invasion in vivo and in vitro, the underlying molecular mechanism remains unknown. Here, we examined the involvement of the LINC complex in radiation‐enhanced cell migration and invasion. A sublethal dose of X‐ray radiation promoted human breast cancer MDA‐MB‐231 cell migration and invasion, whereas carbon ion beam radiation suppressed these processes in a dose‐dependent manner. Depletion of SUN1 and SUN2 significantly suppressed X‐ray‐enhanced cell migration and invasion. Moreover, depletion or overexpression of each SUN1 splicing variant revealed that SUN1_888 containing 888 amino acids of SUN1 but not SUN1_916 was required for X‐ray‐enhanced migration and invasion. In addition, the results suggested that X‐ray irradiation affected the expression level of SUN1 splicing variants and a SUN protein binding partner, nesprins. Taken together, our findings supported that the LINC complex contributed to photon‐enhanced cell migration and invasion.

SPOP, speckle-type POZ protein is a substrate adaptor protein of the Cullin-3/RING ubiquitin E3 complex. The spop gene is the most commonly point mutated in human primary prostate cancers, but the pathological contribution of the SPOP mutations remains unclear. In this study, we investigated several known factors that are critical in the DNA--protein cross-link repair process. The depletion of SPOP or overexpression of a prostate cancer-associated SPOP mutant, F133V, in androgen receptor-positive prostate cancer cells increased the amount of topoisomerase 2A (TOP2A) in the nuclei together with the increased amount of γH2AX, an indication of DNA breaks. Tyrosyl-DNA phosphodiesterases (TDPs) and an endo/exonuclease MRE11 are enzymes that liberate TOP2A from the TOP2A-DNA cleavage complex, and thus is essential for the completion of the DNA repair process. We found that the amount of TDP1 and TDP2 was decreased in SPOP-depleted cells, and that of TDP2 and MRE11 was decreased in F133V-overexpressing cells. These results suggest that the F133V mutant exerts dominant-negative and gain-of-function effects in down-regulation of TDP2 and MRE11, respectively. We conclude that SPOP is involved in the DNA-protein cross-link repair process through the elimination of TOP2A from the TOP2A cleavage complex, which may contribute to the genome stability.

A supervised machine learning algorithm, which is qualified for image classification and analyzing similarities, is based on multiple discriminative morphological features that are automatically assembled during the learning processes. The algorithm is suitable for population-based analysis of images of biological materials that are generally complex and heterogeneous. Here we used the algorithm wndchrm to quantify the effects on nucleolar morphology of the loss of the components of nuclear envelope in a human mammary epithelial cell line. The linker of nucleoskeleton and cytoskeleton (LINC) complex, an assembly of nuclear envelope proteins comprising mainly members of the SUN and nesprin families, connects the nuclear lamina and cytoskeletal filaments. The components of the LINC complex are markedly deficient in breast cancer tissues. We found that a reduction in the levels of SUN1, SUN2, and lamin A/C led to significant changes in morphologies that were computationally classified using wndchrm with approximately 100% accuracy. In particular, depletion of SUN1 caused nucleolar hypertrophy and reduced rRNA synthesis. Further, wndchrm revealed a consistent negative correlation between SUN1 expression and the size of nucleoli in human breast cancer tissues. Our unbiased morphological quantitation strategies using wndchrm revealed an unexpected link between the components of the LINC complex and the morphologies of nucleoli that serves as an indicator of the malignant phenotype of breast cancer cells.

The primary functions of the nuclear envelope are to isolate the nucleoplasm and its contents from the cytoplasm as well as maintain the spatial and structural integrity of the nucleus. The nuclear envelope also plays a role in the transfer of various molecules and signals to and from the nucleus. To reach the nucleus, an extracellular signal must be transmitted across three biological membranes: the plasma membrane, as well as the inner and outer nuclear membranes. While signal transduction across the plasma membrane is well characterized, signal transduction across the nuclear envelope, which is essential for cellular functions such as transcriptional regulation and cell cycle progression, remains poorly understood. As a physical entity, the nuclear envelope, which contains more than 100 proteins, functions as a binding scaffold for both the cytoskeleton and the nucleoskeleton, and acts in mechanotransduction by relaying extracellular signals to the nucleus. Recent results show that the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, which is a conserved molecular bridge that spans the nuclear envelope and connects the nucleoskeleton and cytoskeleton, is also capable of transmitting information bidirectionally between the nucleus and the cytoplasm. This short review discusses bidirectional signal transduction across the nuclear envelope, with a particular focus on mechanotransduction.

The linker of nucleoskeleton and cytoskeleton (LINC) complex, containing the proteins SUN and nesprin, is the fundamental structural unit of the nuclear envelope. The neoplastic-based regulation of the LINC complex in cancer tissues has become increasingly recognized in recent years, including the altered expression, somatic mutation, and methylation of genes. However, precisely how mutations and deregulated expression of the LINC complex contribute to the pathogenic mechanisms of tumorigenesis remain to be elucidated, mainly because of several technical difficulties. First, both the SUN and SYNE (encoding nesprin) genes give rise to a vast number of splicing variants. Second, immunoprecipitation experiments of endogenous SUN and nesprin proteins are difficult owing to the lack of suitable reagents as well as the limited solubility of these proteins in mild extraction conditions. Here, we describe three protocols to investigate these aspects: (1) immunohistochemistry to determine the expression levels and localization of the LINC complex in cancer tissue, (2) detection of SUN1 splicing variants at the mRNA level, and (3) detection of SUN1 splicing variants and binding partners at the protein level.

Cancer cells exhibit a variety of features indicative of atypical nuclei. However, the molecular mechanisms underlying these phenomena remain to be elucidated. The linker of nucleoskeleton and cytoskeleton (LINC) complex, a nuclear envelope protein complex consisting mainly of the SUN and nesprin proteins, connects nuclear lamina and cytoskeletal filaments and helps to regulate the size and shape of the nucleus. Using immunohistology, we found that a nuclear lamina component, lamin A/C and all of the investigated LINC complex components, SUN1, SUN2, and nesprin-2, were downregulated in human breast cancer tissues. In the majority of cases, we observed lower expression levels of these analytes in samples' cancerous regions as compared to their cancer-associated noncancerous regions (in cancerous regions, percentage of tissue samples exhibiting low protein expression: lamin A/C, 85% [n = 73]; SUN1, 88% [n = 43]; SUN2, 74% [n = 43]; and nesprin-2, 79% [n = 53]). Statistical analysis showed that the frequencies of recurrence and HER2 expression were negatively correlated with lamin A/C expression (P

The linker of nucleoskeleton and cytoskeleton (LINC) complex, containing the proteins SUN and nesprin, is the fundamental structural unit of the nuclear envelope. The neoplastic-based regulation of the LINC complex in cancer tissues has become increasingly recognized in recent years, including the altered expression, somatic mutation, and methylation of genes. However, precisely how mutations and deregulated expression of the LINC complex contribute to the pathogenic mechanisms of tumorigenesis remain to be elucidated, mainly because of several technical difficulties. First, both the SUN and SYNE (encoding nesprin) genes give rise to a vast number of splicing variants. Second, immunoprecipitation experiments of endogenous SUN and nesprin proteins are difficult owing to the lack of suitable reagents as well as the limited solubility of these proteins in mild extraction conditions. Here, we describe three protocols to investigate these aspects: (1) immunohistochemistry to determine the expression levels and localization of the LINC complex in cancer tissue, (2) detection of SUN1 splicing variants at the mRNA level, and (3) detection of SUN1 splicing variants and binding partners at the protein level.

The LINC complex is a multifunctional protein complex that is involved in various processes at the nuclear envelope, such as nuclear migration, mechanotransduction and chromatin tethering in the meiotic phase. However, it remains unknown how these functions are regulated in different cell contexts. An inner nuclear membrane component of the LINC complex, SUN1, is ubiquitously expressed. The human SUN1 gene produces over 10 variants by alternative splicing. Although functions of SUN1 are relatively well characterized, functional differences among SUN1 splice variants are poorly characterized. LINC complex components are associated with a wide range of human diseases; therefore, it is important to understand the functional diversity among SUN1 splice variants. Here, we identified a novel human SUN1 splice variant, SUN1_888. overexpression of the SUN1 splice variants, SUN1_888 or SUN1_785, but not the predominant isoform, SUN1_916, activated directional cell migration. Knockdown of SUN1_888 suppressed cell migration; in contrast depletion of SUN1_916 activated cell migration. In addition, all of investigated SUN1 splicing variants rescued cell migration in SUN1 knock out cell. These results indicate that redundant and non-redundant functions of SUN1 splice variant in directional cell migration and suggest that variable LINC complexes with distinct task may exit. Furthermore, in contrast to previous studies, we showed association between SUN1 and B-type lamins. Interestingly, B-type lamin preferentially interacts with SUN1 but not SUN2. These results suggest that tissue-specific SUN1 variants variably interact with nucleoplasmic partners and allow variable assembly of LINC complexes that can be assigned to distinct tasks.

Amphiregulin (AREG), an EGF family protein, is synthesized as a type I transmembrane precursor (proAREG) and expressed on the cell surface with an extracellular EGF-like domain and an intracellular short cytoplasmic tail. The ectodomain shedding yields a soluble EGF receptor ligand (soluble AREG) which binds to EGF receptor (EGFR) and concomitantly induces migration of unshed proAREG from the plasma membrane to the nuclear envelope (NE). AREG is known to play a potential role in breast cancer and has been intensively investigated as an EGF receptor ligand, while the function of the NE-localized proAREG remains unknown. In this study we used a truncated mutant that mimics NE-localized proAREG without shedding stimuli to discriminate between the functions of NE-localized and plasma membrane-localized proAREG and demonstrate that NE-localized proAREG activates breast cancer cell migration, but suppresses cell growth. Moreover, the present study shows that induction of cell migration by NE-localized proAREG does not require the extracellular growth factor domain or EGF receptor function. Collectively these data demonstrate a novel function mediated by the intracellular domain of proAREG and suggest a significant role for NE-localized proAREG in driving human breast cancer progression.

Objective: The knowledge on the association between platelet-derived growth factor (PDGF) signaling and epithelial cancers is scarce, although overexpression of PDGF and PDGF receptors has been reported in some human mesenchymal tumors. Thus, we studied the effect of PDGF on breast cancer cells in vitro and the distribution of PDGF in breast cancer tissues. Methods: The effect of PDGF-BB on breast cancer cells was assessed by Western blotting, immunofluorescence, WST and 5-bromo-2-deoxyuridine incorporation experiments. PDGF-B and β-catenin expression was investigated in breast cancer tissues by immunohistochemistry. Results: PDGF-BB induces β-catenin expression in breast cancer cells, and immunohistochemically the distribution of PDGF-B was similar to β-catenin in breast cancer cells. PDGF-B-positive cancer cells were more frequent in cases of ductal carcinoma in situ (87.5%) than invasive carcinoma (61.2%). In addition, PDGF-B staining was stronger in intraductal than invasive cancer cells. PDGF-BB tended to induce nuclear translocation of β-catenin, cell proliferation and DNA incorporation in MDA-MB231 cells, while these results were not found in MCF-7 cells. Conclusion: Our results suggest that PDGF-BB regulates protein expression of β-catenin and is associated with cancer cell behavior.

Amphiregulin (AR), a member of the EGF family, is synthesized as a type I transmembrane protein precursor (proAR) and expressed on the cell surface. Shedding of proAR yields a transmembrane-cytoplasmic fragment (AR-CTF), as well as a soluble AR. Here we demonstrate that the proAR-shedding stimuli trigger endocytosis of both AR-CTF and un-shed proAR. ProAR translocates from the plasma membrane to the inner nuclear membrane, whereas AR-CTF is translocated to the lysosome via retrograde membrane trafficking. Nuclear envelope localization of proAR involves truncation of the C-terminus, which subsequently activates the ER-retrieval signal. The truncated form of proAR interacts with A-type lamin and is retained at the inner nuclear membrane. Heterochromatin formation is then induced and global transcription is transiently suppressed. This study gives new insight into epigenetic chromatin organization in mammalian cells: a plasma-membrane-anchored growth factor is targeted to the inner nuclear membrane where it participates in dynamic chromatin organization and control of transcription.

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein (proHB-EGF) and expressed on the cell surface. The ectodomain shedding of proHB-EGF at the extracellular region on the plasma membrane yields a soluble EGF receptor ligand and a transmembrane-cytoplasmic fragment (HB-EGF-CTF). The cytoplasmic domain of proHB-EGF (HB-EGF-cyto) interacts with transcriptional repressors to reverse their repressive activities. However, how HB-EGF-cyto accesses transcriptional repressors is yet unknown. The present study demonstrates that, after exposure to shedding stimuli, both HB-EGF-CTF and unshed proHB-EGF translocate to the nuclear envelope. Immunoelectron microscopy and digitonin-permeabilized cells showed that HB-EGF-cyto signals are at the inner nuclear membrane. A short sequence element within the HB-EGF-cyto allows a transmembrane protein to localize to the nuclear envelope. The dominant-active form of Rab5 and Rab11 suppressed nuclear envelope targeting. Collectively, these data demonstrate that membrane-anchored HB-EGF is targeted to the inner nuclear membrane via a retrograde membrane trafficking pathway.

The epidermal growth factor (EGF) family and the EGF receptor (EGFR, ErbB) tyrosine kinase family have been spearheading the studies of signal transduction events that determine cell fate and behavior in vitro and in vivo. The EGFR family and their signaling pathways are giving us tremendous advantages in developing fascinating molecular target strategies for cancer therapy. Currently, two important types of EGFR inhibitors are in clinical use: neutralizing antibodies of EGFR or ErbB2, and synthetic small compounds of tyrosine kinase inhibitors designed for receptors. On the other hand, basic research of the EGF family ligands presents new challenges as membrane-anchored growth factors. All members of the EGF family have important roles in development and diseases and are shed from the plasma membrane by metalloproteases. The ectodomain shedding of the ligands has emerged as a critical component in the functional transactivation of EGFRs in interreceptor cross-talk in response to various shedding stimulants such as G-protein coupled receptor agonists, growth factors, cytokines, and various physicochemical stresses. Among the EGFR-ligands, heparin-binding EGF-like growth factor (HB-EGF) is a prominent ligand in our understanding of the pathophysiological roles of ectodomain shedding in cancer, wound healing, cardiac diseases, etc. Here we focus on ectodomain shedding of the EGF family ligands, especially HB-EGF by disintegrin and metalloproteases, which are not only key events of receptor cross talk, but also novel intercellular signaling by their carboxy-terminal fragments to regulate gene expression directly.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family, is synthesized as a type I transmembrane precursor (pro-HB-EGF). Ectodomain shedding of pro-HB-EGF yields an amino-terminal soluble ligand of EGF receptor (HB-EGF) and a carboxyl-terminal fragment (HB-EGF-CTF) consisting of the transmembrane and cytoplasmic domains. We previously showed that the HB-EGF-CTF translocates from the plasma membrane to the nucleus and plays a role as a signaling molecule. Immunoprecipitation showed that HB-EGF-CTF can associate with Bcl6, a transcriptional repressor in mammalian cells. A glutathione S-transferase pulldown assay revealed that HB-EGF-CTF interacted efficiently with zinc fingers 4-6 of Bcl6. A luciferase reporter assay showed that the nuclear translocation of HB-EGF-CTF following shedding reversed transcriptional repression of cyclin D2 by Bcl6. Additionally, the level of cyclin D2 protein increased and Bcl6 interaction with the cyclin D2 promoter decreased in parallel with the shedding of pro-HB-EGF at all endogenous levels. These findings suggest that HB-EGF-CTF is a potent regulator of gene expression via its interaction with the transcriptional repressor Bcl6.

Ran is a nuclear Ras-like GTPase that is required for various nuclear events including the bi-directional transport of proteins and ribonucleoproteins through the nuclear pore complex, spindle formation, and reassembly of the nuclear envelope. One of the key regulators of Ran is RanGAP1, a Ran specific GTPase activating protein. The question of whether a mechanism exists for controlling nucleocytoplasmic transport through the regulation of RanGAP1 activity continues to be debated. Here we show that RanGAP1 is phosphorylated in vivo and in vitro. Serine-358 (358S) was identified as the major phosphorylation site, by MALDI-TOF-MS spectrometry. Site directed mutagenesis at this position abolished the phosphorylation. Experiments using purified recombinant kinase and specific inhibitors such as DRB and apigenin strongly suggest that casein kinase II (CK2) is the responsible kinase. Although the phosphorylation of 358S of RanGAP1 did not significantly alter its GAP activity, the phosphorylated wild type RanGAP1, but not a mutant harboring a mutation at the phosphorylation site 358S, efficiently formed a stable ternary complex with Ran and RanBP1 in vivo, suggesting that the 358S phosphorylation of RanGAP1 affects the Ran system.

Genome-wide aberrant histone modifications are present in a wide range of cancers, and they are associated with carcinogenesis and cancer progression. Aberrant histone modification patterns affect transcriptional regulation, chromosome stability, chromatin structure, chromatin remodeling, and DNA methylation; furthermore, these patterns can predict clinical outcome in many types of cancer. The main cause of poor clinical outcome is metastasis, which is strongly associated with tissue invasion at the primary tumor site. Invasion of cancer cells into surrounding tissue and the vasculature is an important initial step in tumor metastasis, and cell migration is a critical requirement for metastasis. Here, we describe the advantages of detecting global histone modifications by immunohistochemical analysis and provide a collection of protocols for assaying cell migration, invasion, and cell-extracellular matrix adhesion in vitro.

Genome-wide aberrant histone modifications are present in a wide range of cancers, and they are associated with carcinogenesis and cancer progression. Aberrant histone modification patterns affect transcriptional regulation, chromosome stability, chromatin structure, chromatin remodeling, and DNA methylation; furthermore, these patterns can predict clinical outcome in many types of cancer. The main cause of poor clinical outcome is metastasis, which is strongly associated with tissue invasion at the primary tumor site. Invasion of cancer cells into surrounding tissue and the vasculature is an important initial step in tumor metastasis, and cell migration is a critical requirement for metastasis. Here, we describe the advantages of detecting global histone modifications by immunohistochemical analysis and provide a collection of protocols for assaying cell migration, invasion, and cell-extracellular matrix adhesion in vitro.

A small GTPase Ran is a key regulator for active nuclear transport. In immunoblotting analysis, a monoclonal antibody against recombinant human Ran, designated ARAN1, was found to recognize an epitope in the COOH-terminal domain of Ran. In a solution binding assay, ARAN1 recognized Ran when complexed with importin β, transportin, and CAS, but not the Ran-GTP or the Ran-GDP alone, indicating that the COOH-terminal domain of Ran is exposed via its interaction with importin β–related proteins. In addition, ARAN1 suppressed the binding of RanBP1 to the Ran–importin β complex. When injected into the nucleus of BHK cells, ARAN1 was rapidly exported to the cytoplasm, indicating that the Ran–importin β–related protein complex is exported as a complex from the nucleus to the cytoplasm in living cells. Moreover, ARAN1, when injected into the cultured cells induces the accumulation of endogenous Ran in the cytoplasm and prevents the nuclear import of SV-40 T-antigen nuclear localization signal substrates. From these findings, we propose that the binding of RanBP1 to the Ran–importin β complex is required for the dissociation of the complex in the cytoplasm and that the released Ran is recycled to the nucleus, which is essential for the nuclear protein transport.

The active transport of proteins into and out of the nucleus is mediated by specific signals, the nuclear localization signal (NLS) and nuclear export signal (NES), respectively. The best characterized NLS is that of the SV40 large T antigen, which contains a cluster of basic amino acids. The NESs were first identified in the protein kinase inhibitor (PKI) and HIV Rev protein, which are rich in leucine residues. The SV40 T-NLS containing transport substrates are carried into the nucleus by an importin alpha/beta heterodimer. Importin alpha recognizes the NLS and acts as an adapter between the NLS and importin beta, whereas importin beta interacts with importin alpha bound to the NLS, and acts as a carrier of the NLS/importin alpha/beta trimer. It is generally thought that importin alpha and beta are part of a large protein family. The leucine rich NES-containing proteins are exported from the nucleus by one of the importin beta family molecules, CRM1/exportin 1. A Ras-like small GTPase Ran plays a crucial role in both import/export pathways and determines the directionality of nuclear transport. It has recently been demonstrated in living cells that Ran actually shuttles between the nucleus and the cytoplasm and that the recycling of Ran is essential for the nuclear transport. Furthermore, it has been shown that nuclear transport factor 2 (NTF2) mediates the nuclear import of RanGDP. This review largely focuses on the issue concerning the functional divergence of importin alpha family molecules and the role of Ran in nucleocytoplasmic protein transport.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:45:14Z No. of bitstreams: 1 S2175-78602012000200008.pdf: 2133434 bytes, checksum: 80366a942ce657fec6da75149670fd4b (MD5) Made available in DSpace on 2013-08-22T18:45:14Z (GMT). No. of bitstreams: 1 S2175-78602012000200008.pdf: 2133434 bytes, checksum: 80366a942ce657fec6da75149670fd4b (MD5) Previous issue date: 2012-06-01 Made available in DSpace on 2013-09-30T18:48:09Z (GMT). No. of bitstreams: 2 S2175-78602012000200008.pdf: 2133434 bytes, checksum: 80366a942ce657fec6da75149670fd4b (MD5) S2175-78602012000200008.pdf.txt: 73902 bytes, checksum: a9d0f89a23ea076360fd2d3fb6a110e1 (MD5) Previous issue date: 2012-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:57:13Z No. of bitstreams: 2 S2175-78602012000200008.pdf: 2133434 bytes, checksum: 80366a942ce657fec6da75149670fd4b (MD5) S2175-78602012000200008.pdf.txt: 73902 bytes, checksum: a9d0f89a23ea076360fd2d3fb6a110e1 (MD5) Made available in DSpace on 2014-05-20T13:57:13Z (GMT). No. of bitstreams: 2 S2175-78602012000200008.pdf: 2133434 bytes, checksum: 80366a942ce657fec6da75149670fd4b (MD5) S2175-78602012000200008.pdf.txt: 73902 bytes, checksum: a9d0f89a23ea076360fd2d3fb6a110e1 (MD5) Previous issue date: 2012-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Amostragem florística foi feita em áreas da Reserva Biológica Municipal da Serra do Japi, Jundiaí, Estado de São Paulo, incluíndo fisionomias de floresta semidecídua montana, classificada como parte das formações da Mata Atlântica. Foram encontradas 661 espécies em 129 famílias, as dez fanerogâmicas mais diversas foram: Asteraceae (56 espécies), Fabaceae (37), Rubiaceae (29), Poaceae (25), Solanaceae (24), Cyperaceae (19), Euphorbiaceae (19), Orchidaceae (19), Melastomataceae (17), e Piperaceae (17). O acréscimo de 322 espécies adicionais compiladas de listagens florísticas suplementares eleva, para a Serra do Japi em geral, o número registrado a 976 espécies em 141 famílias. Comparada a outros levantamentos florísticos na Mata Atlântica, esses resultados demonstram a diversidade florística da área estudada e ressaltam o valor da preservação da vegetação nessa região altamente urbanizada. Floristic sampling was carried out in Serra do Japi Biological Reserve, Jundiaí, state of São Paulo, Brazil, including physiognomies of semideciduous montane forest, a type of forest formation within the Atlantic Forest domain. Six hundred and sixty one species distributed in 129 families were recorded; the ten most diverse phanerogam families were: Asteraceae (56 species), Fabaceae (37), Rubiaceae (29), Poaceae (25), Solanaceae (24), Cyperaceae (19), Euphorbiaceae (19), Orchidaceae (19), Melastomataceae (17), and Piperaceae (17). The addition of 322 species from other floristic surveys increased the number of species of Serra do Japi to 976, distributed in 141 families. Compared to other floristic surveys carried out in the Atlantic Forest, our results highlight the floristic diversity of the study area and the importance of preserving the vegetation of this highly urbanized region. Universidade Estadual Paulista - UNESP Instituto de Biociências de Rio Claro Depto. Botânica Research Productivity Fellowship CNPq Universidade Estadual Paulista Undergraduate Program in Biological Sciences Instituto de Biociências de Rio Claro Universidade Estadual Paulista - UNESP Instituto de Biociências de Rio Claro Depto. Botânica Universidade Estadual Paulista Undergraduate Program in Biological Sciences Instituto de Biociências de Rio Claro

RNA polymerase II is responsible for transcription of most eukaryotic genes, but, despite exhaustive analysis, little is known about how it transcribes natural templates in vivo. We studied polymerase dynamics in living Chinese hamster ovary cells using an established line that expresses the largest (catalytic) subunit of the polymerase (RPB1) tagged with the green fluorescent protein (GFP). Genetic complementation has shown this tagged polymerase to be fully functional. Fluorescence loss in photobleaching (FLIP) reveals the existence of at least three kinetic populations of tagged polymerase: a large rapidly-exchanging population, a small fraction resistant to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) but sensitive to a different inhibitor of transcription (i.e. heat shock), and a third fraction sensitive to both inhibitors. Quantitative immunoblotting shows the largest fraction to be the inactive hypophosphorylated form of the polymerase (i.e. IIA). Results are consistent with the second (DRB-insensitive but heat-shock-sensitive) fraction being bound but not engaged, while the third (sensitive to both DRB and heat shock) is the elongating hyperphosphorylated form (i.e. IIO).

Publisher Summary The dynamics of proteins can be monitored in living cells after tagging them with the green fluorescent protein (GFP). A construct encoding GFP fused to the protein of interest is expressed in a cell, so the resulting fluorescent hybrid can be seen directly. Mutant GFPs with altered fluorescence are available (e.g., enhanced cyan and yellow fluorescent proteins—ECFPs and EYFPs), and one—PAGFP—is photoactivated by irradiation with 413-nm light so its fluorescence (488-nm excitation) increases 100-fold. Therefore, it is possible to monitor histone and polymerase dynamics in real time, using these GFP tags and photobleaching techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). This chapter presents the studies on the dynamics of the core histones and RNA polymerase II to illustrate these techniques.

A classical nuclear localization signal (NLS)-containing protein is transported into the nucleus via the formation of a NLS-substrate/importin alpha/beta complex. In this study, we found that importin alpha migrated into the nucleus without the addition of importin beta, Ran or any other soluble factors in an in vitro transport assay. A mutant importin alpha lacking the importin beta-binding domain efficiently entered the nucleus. Competition experiments showed that this import pathway for importin alpha is distinct from that of importin beta. These results indicate that importin alpha alone can enter the nucleus via a novel pathway in an importin beta- and Ran-independent manner. Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae. Moreover, the import rate of importin alpha differed among individual nuclei of permeabilized cells, as demonstrated by time-lapse experiments. This heterogeneous nuclear accumulation of importin alpha was affected by the addition of ATP, but not ATPgammaS. These results suggest that the nuclear import machinery for importin alpha at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis.

U1A is a component of the uracil-rich small nuclear ribonucleoprotein. The molecular mechanism of nuclear import of U1A was investigated in vivo and in vitro. When recombinant deletion mutants of U1A are injected into the BHK21 cell cytoplasm, the nuclear localization signal (NLS) of U1A is found in the N-terminal half of the central domain (residues 100-144 in mouse U1A). In an in vitro assay, it was found that the U1A-NLS accumulated in only a portion of the nuclei in the absence of cytosolic extract. In contrast, the addition of importin alpha/beta and Ran induced the uniform nuclear accumulation of U1A-NLS in all cells. Furthermore, U1A was found to bind the C-terminal portion of importin alpha. In addition, the in vitro nuclear migration of full-length U1A was found to be exclusively dependent on importin alpha/beta and Ran. Moreover, in living cells, the full-length U1A accumulated in the nucleus in a Ran-dependent manner, and nuclear accumulation was inhibited by the importin beta binding domain of importin alpha. These results suggest that the nuclear import of U1A is mediated by at least two distinct pathways, an importin alpha/beta and Ran-dependent and an -independent pathway in permeabilized cells, and that the latter pathway may be suppressed in intact cells.

We previously reported that the nuclear import of substrates containing SV40 T antigen nuclear localization signal (NLS) was suppressed in a temperature-sensitive RCC1 mutant cell line, tsBN2, at nonpermissive temperature. Moreover, it was shown that import into wild type BHK21 cell-derived nuclei gradually decreased in heterokaryons between the tsBN2 and BHK21 cells, although the BHK21 nuclei retained wild type RCC1 and should contain RanGTP (Tachibana et al., 1994). In this study, it was found that in the heterokaryons cultured at non-permissive temperature, endogenous importin alpha was not detected immunocytochemically in the cytoplasm or BHK21 nuclei but only in the tsBN2 nuclei, suggesting that importin alpha cannot be exported from the RCC1-depleted nuclei. In fact, importin alpha microinjected into the nucleus of tsBN2 cells at non-permissive temperature remained in the nucleus. These results strongly support the hypothesis that the recycling of importin alpha from the nucleus requires nuclear RanGTP. Moreover, it was found that cytoplasmic injection of importin alpha restored the import of SV40 T-NLS substrates in the BHK21 nuclei but not the tsBN2 nuclei in the heterokaryons. This indicates that the decrease of importin alpha from the cytoplasm in the heterokaryons leads to a suppression of the efficiency of nuclear import of the T-NLS substrate and provides support for the view that nuclear RanGTP is essential for the nuclear entry of the substrates.

Heparin-binding epidermal growth factor (EGF)- like growth factor (HB-EGF) is synthesized in the ER, transported along the exocytic pathway, and expressed on the plasma membrane as a type I transmembrane protein. Upon extracellular stimulation, HB-EGF, either proHB-EGF or the shed form HB-EGF-CTF, undergoes endocytosis and is then transported retrogradely to the ER. In this study, we showed the essential contribution of the short cytoplasmic tail of HB-EGF (HB-EGF-cyto) to the bidirectional intracellular trafficking between the ER and plasma membrane and revealed several critical amino acids residues that are responsible for internalization from the plasma membrane and ER targeting. We suggest that these anterograde and retrograde sorting signals within HB-EGF-cyto are strictly regulated by protein modification and conformation., Highlights ▸ The cytoplasmic tail of HB-EGF contributes to bidirectional intracellular trafficking. ▸ The carboxyl-terminal five amino acids are indispensable for internalization. ▸ Amino acids S207 and K201 play essential roles in retrograde transport to the ER. ▸ Sorting signals in HB-EGF are regulated by protein modification and conformation. ▸ The extracellular domain of proHB-EGF is not required for intracellular trafficking.

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.

Using an in vivo assay system, nuclear import ability in individual cells was determined by examining the nuclear import rate. It was found that when a small (not excess) amount of SV40 T-NLS peptides was co-injected, the nuclear import rate of SV40 T-NLS-containing substrates apparently increased. This up-regulation was reproduced by the co-injection of peptides containing bipartite type NLS of CBP80, but not mutated non-functional NLS peptides, which suggests that these phenomena are specific for functional NLSs. It was further shown that although, in growth-arrested cells, the nuclear import rate was down-regulated compared to growing cells, the elevation of the functional import rate by co-injected NLS peptides reached the same level as in proliferating cells. This up-regulation was abolished by the addition of a protein kinase inhibitor, staurosporine. These results suggest that although potential nuclear import ability does not vary in each cell, the rate of nuclear import may be controlled by the amount of karyophilic proteins, which need to be carried into the nucleus from the cytoplasm, possibly via an NLS-dependent phosphorylation reaction.