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Cloning and Characterization of Sf9 Cell Lamin and the Lamin Conformational Changes during Autographa californica multiple nucleopolyhedrovirus Infection
Abstract:At present, the details of lamina alterations after baculovirus infection remain elusive. In this study, a lamin gene in the Sf9 cell line of Spodoptera frugiperda was cloned. The open reading frame (orf) of the Sf9 lamin was 1860 bp and encoded a protein with a molecular weight of 70 kDa. A transfection assay with a red fluorescence protein (rfp)-lamin fusion protein indicated that Sf9 lamin was localized in the nuclear rim. Transmission electron microscopy observations indicated that Autographa californica m… Show more
“…3A ). As the total amount of lamin B did not increase during the course of infection according to the Western blot results and a recent study 32 , these data implied a partial disruption of the nuclear lamina and redistribution of the disassembled GFP-lamin B within the nuclei during baculovirus infection. Figure 3 Subcellular localization of GFP-lamin B in Sf9-L cells.…”
Section: Resultssupporting
confidence: 73%
“…Wei et al . 32 detected lamin localization beginning at 48 h p.i., and therefore, diffuse localization of lamins may not have been observed. In addition, the authors did not observe lamin redistribution within the nuclei of infected cells, unlike the results obtained in our study.…”
Section: Discussionmentioning
confidence: 96%
“…During the preparation of this manuscript, Wei et al . 32 cloned a lamin gene in the Sf9 cell line. We compared the amino acid sequence of Sf9 lamin with Drosophila lamin B and found that all the experimentally confirmed phosphorylation sites for Drosophila lamin B are fully conserved between these two lamins (data not shown).…”
Section: Discussionmentioning
confidence: 99%
“…Wei et al . 32 also described alterations to the Sf9 nuclear lamina during AcMNPV infection based on immunofluorescence analyses. However, they did not find remarkable structure alternations.…”
Section: Discussionmentioning
confidence: 99%
“…This inconsistency is potentially due to the different methods used for fluorescence detection in the two studies: lamin B localization was observed by detecting immunofluorescence associated with the ADL67 antibody, which binds lamin B, in the report by Wei et al . 32 , but localization was monitored based on GFP autofluorescence in the present analysis. GFP autofluorescence intensity may be stronger than that of the immunofluorescent signal associated with the ADL67 antibody.…”
Baculovirus nucleocapsids egress from the nucleus primarily via budding at the nuclear membrane. The nuclear lamina underlying the nuclear membrane represents a substantial barrier to nuclear egress. Whether the nuclear lamina undergoes disruption during baculovirus infection remains unknown. In this report, we generated a clonal cell line, Sf9-L, that stably expresses GFP-tagged Drosophila lamin B. GFP autofluorescence colocalized with immunofluorescent anti-lamin B at the nuclear rim of Sf9-L cells, indicating GFP-lamin B was incorporated into the nuclear lamina. Meanwhile, virus was able to replicate normally in Sf9-L cells. Next, we investigated alterations to the nuclear lamina during baculovirus infection in Sf9-L cells. A portion of GFP-lamin B localized diffusely at the nuclear rim, and some GFP-lamin B was redistributed within the nucleus during the late phase of infection, suggesting the nuclear lamina was partially disrupted. Immunoelectron microscopy revealed associations between GFP-lamin B and the edges of the electron-dense stromal mattes of the virogenic stroma, intranuclear microvesicles, and ODV envelopes and nucleocapsids within the nucleus, indicating the release of some GFP-lamin B from the nuclear lamina. Additionally, GFP-lamin B phosphorylation increased upon infection. Based on these data, baculovirus infection induced lamin B phosphorylation and disruption of the nuclear lamina.
“…3A ). As the total amount of lamin B did not increase during the course of infection according to the Western blot results and a recent study 32 , these data implied a partial disruption of the nuclear lamina and redistribution of the disassembled GFP-lamin B within the nuclei during baculovirus infection. Figure 3 Subcellular localization of GFP-lamin B in Sf9-L cells.…”
Section: Resultssupporting
confidence: 73%
“…Wei et al . 32 detected lamin localization beginning at 48 h p.i., and therefore, diffuse localization of lamins may not have been observed. In addition, the authors did not observe lamin redistribution within the nuclei of infected cells, unlike the results obtained in our study.…”
Section: Discussionmentioning
confidence: 96%
“…During the preparation of this manuscript, Wei et al . 32 cloned a lamin gene in the Sf9 cell line. We compared the amino acid sequence of Sf9 lamin with Drosophila lamin B and found that all the experimentally confirmed phosphorylation sites for Drosophila lamin B are fully conserved between these two lamins (data not shown).…”
Section: Discussionmentioning
confidence: 99%
“…Wei et al . 32 also described alterations to the Sf9 nuclear lamina during AcMNPV infection based on immunofluorescence analyses. However, they did not find remarkable structure alternations.…”
Section: Discussionmentioning
confidence: 99%
“…This inconsistency is potentially due to the different methods used for fluorescence detection in the two studies: lamin B localization was observed by detecting immunofluorescence associated with the ADL67 antibody, which binds lamin B, in the report by Wei et al . 32 , but localization was monitored based on GFP autofluorescence in the present analysis. GFP autofluorescence intensity may be stronger than that of the immunofluorescent signal associated with the ADL67 antibody.…”
Baculovirus nucleocapsids egress from the nucleus primarily via budding at the nuclear membrane. The nuclear lamina underlying the nuclear membrane represents a substantial barrier to nuclear egress. Whether the nuclear lamina undergoes disruption during baculovirus infection remains unknown. In this report, we generated a clonal cell line, Sf9-L, that stably expresses GFP-tagged Drosophila lamin B. GFP autofluorescence colocalized with immunofluorescent anti-lamin B at the nuclear rim of Sf9-L cells, indicating GFP-lamin B was incorporated into the nuclear lamina. Meanwhile, virus was able to replicate normally in Sf9-L cells. Next, we investigated alterations to the nuclear lamina during baculovirus infection in Sf9-L cells. A portion of GFP-lamin B localized diffusely at the nuclear rim, and some GFP-lamin B was redistributed within the nucleus during the late phase of infection, suggesting the nuclear lamina was partially disrupted. Immunoelectron microscopy revealed associations between GFP-lamin B and the edges of the electron-dense stromal mattes of the virogenic stroma, intranuclear microvesicles, and ODV envelopes and nucleocapsids within the nucleus, indicating the release of some GFP-lamin B from the nuclear lamina. Additionally, GFP-lamin B phosphorylation increased upon infection. Based on these data, baculovirus infection induced lamin B phosphorylation and disruption of the nuclear lamina.
Cellular senescence is the arrest of normal cell division. Oncogenic genes and oxidative stress, which cause genomic DNA damage and generation of reactive oxygen species, lead to cellular senescence. The senescence-associated secretory phenotype is a distinct feature of senescence. Senescence is normally involved in the embryonic development. Senescent cells can communicate with immune cells to invoke an immune response. Senescence emerges during the aging process in several tissues and organs. In fact, increasing evidence shows that cellular senescence is implicated in aging-related diseases, such as nonalcoholic fatty liver disease, obesity and diabetes, pulmonary hypertension, and tumorigenesis. Cellular senescence can also be induced by microbial infection. During cellular senescence, several signaling pathways, including those of p53, nuclear factor-κB (NF-κB), mammalian target of rapamycin, and transforming growth factor-beta, play important roles. Accumulation of senescent cells can trigger chronic inflammation, which may contribute to the pathological changes in the elderly. Given the variety of deleterious effects caused by cellular senescence in humans, strategies have been proposed to control senescence. In this review, we will focus on recent studies to provide a brief introduction to cellular senescence, including associated signaling pathways and pathology.
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