Healthy children are more likely to die of influenza Avirus (IAV) infection than healthy adults. However, little is known about the mechanisms underlying the impact of young age on the development of life-threatening IAV infection. We report increased mortality in juvenile mice compared with adult mice at each infectious dose of IAV. Juvenile mice had sustained elevation of type I IFNs and persistent NLRP3 inflammasome activation in the lungs, both of which were independent of viral titer. Juvenile mice, but not adult mice, had increased MCP-1 levels that remained high even after viral clearance. Importantly, continued production of MCP-1 was associated with persistent recruitment of monocytes to the lungs and prolonged elevation of inflammatory cytokines. Transcriptional signatures of recruited monocytes to the juvenile and adult IAV-infected lungs were assessed by RNA-seq. Genes associated with a proinflammatory signature were upregulated in the juvenile monocytes compared with adult monocytes. Depletion of monocytes with anti-CCR2 Ab decreased type I IFN secretion, NLRP3 inflammasome activation, and lung injury in juvenile mice. This suggests an exaggerated inflammatory response mediated by increased recruitment of monocytes to the lung, and not an inability to control viral replication, is responsible for severe IAV infection in juvenile mice. This study provides insight into severe IAV infection in juveniles and identifies key inflammatory monocytes that may be central to pediatric acute lung injury secondary to IAV.

Influenza A virus (IAV) is a significant cause of life-threatening lower respiratory tract infections in children. Antiviral therapy is the mainstay of treatment, but its effectiveness in this age group has been questioned. In addition, damage inflicted on the lungs by the immune response to the virus may be as important to the development of severe lung injury during IAV infection as the cytotoxic effects of the virus itself. A crucial step in the immune response to IAV is activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and the subsequent secretion of the inflammatory cytokines, interleukin-1β (IL-1β), and interleukin-18 (IL-18). The IAV matrix 2 proton channel (M2) has been shown to be an important activator of the NLRP3 inflammasome during IAV infection. We sought to interrupt this ion channel-mediated activation of the NLRP3 inflammasome through inhibition of NLRP3 or the cytokine downstream from its activation, IL-1β. Using our juvenile mouse model of IAV infection, we show that inhibition of the NLRP3 inflammasome with the small molecule inhibitor, MCC950, beginning 3 days after infection with IAV, improves survival in juvenile mice. Treatment with MCC950 reduces NLRP3 levels in lung homogenates, decreases IL-18 secretion into the alveolar space, and inhibits NLRP3 inflammasome activation in alveolar macrophages. Importantly, inhibition of the NLRP3 inflammasome with MCC950 does not impair viral clearance. In contrast, inhibition of IL-1β signaling with the IL-1 receptor antagonist, anakinra, is insufficient to protect juvenile mice from IAV. Our findings suggest that targeting the NLRP3 inflammasome in juvenile IAV infection may improve disease outcomes in this age group.

Activation of the NLRP3 inflammasome and subsequent maturation of IL‐1β have been implicated in acute lung injury (ALI), which is characterized by inflammation, IL‐1β release, alveolar epithelial cell injury, hyperplasia of alveolar type 2 cells, accumulation of fibroblasts, deposition of collagen, and scar formation. We investigated the role of vimentin, a type III intermediate filament, in this process using three well characterized murine models of ALI which require NLRP3 for inflammasome activation. It was demonstrated that in these ALI models, the pathophysiologic changes are attenuated in Vim−/− mice. We hypothesized that vimentin directly interacts with NLRP3 for normal inflammasome activity. Therefore, we examined, by co‐immunoprecipitation, whether NLRP3 and vimentin interact in a human macrophage cell line, THP‐1, and mouse bone marrow derived macrophages (BMDM). Next, using bio‐layer interferometery we investigated whether NLRP3 and vimentin directly interact. Preliminary results indicated that full length vimentin directly interacts with NLRP3 with a KD around 240 nM. To investigate which region of vimentin interacts with NLRP3 we used peptide arrays. Our results indicate that the vimentin head domain has and a small region of the tail domain interact with NLRP3 while the central rod domain shows very little interaction. Since vimentin has a large number of amino acids which can be phosphorylated, the binding between phosphomimetic vimentin peptides and NLRP3 was also examined. Phosphomimetic peptides of the vimentin head domain inhibit binding to NLRP3 while increased binding was observed in the tail domain when T426D and S430D mutations were examined. To further demonstrate a functional interaction between vimentin and NLRP3, vimentin was deleted from BMDMs using crispr/Cas9 and vimentin domains were expressed in these Vim KO cells using lentiviral infection. These new cell lines were examined for IL‐1β release after activation with LPS and ATP. Additionally, localization of vimentin and vimentin domains near NLRP3 after activation was investigated using proximity ligation assays. Our data strongly indicate that NLRP3 and vimentin directly interact and that this interaction is important for NLRP3 inflammasome activity.Support or Funding InformationThis work was supported by National Heart, Lung, and Blood Institute (HL71643; HL128194)

The type III intermediate filament protein vimentin was once thought to function mainly as a static structural protein in the cytoskeleton of cells of mesenchymal origin. Now, however, vimentin is known to form a dynamic, flexible network that plays an important role in a number of signaling pathways. Here, we describe various methods that have been developed to investigate the cellular functions of the vimentin protein and intermediate filament network, including chemical disruption, photoactivation and photoconversion, biolayer interferometry, soluble bead binding assay, three-dimensional substrate experiments, collagen gel contraction, optical-tweezer active microrheology, and force spectrum microscopy. Using these techniques, the contributions of vimentin to essential cellular processes can be probed in ever further detail.

Activation of the NLRP3 inflammasome and subsequent maturation of IL-1β have been implicated in acute lung injury (ALI), resulting in inflammation and fibrosis. We investigated the role of vimentin, a type III intermediate filament, in this process using three well-characterized murine models of ALI known to require NLRP3 inflammasome activation. We demonstrate that central pathophysiologic events in ALI (inflammation, IL-1β levels, endothelial and alveolar epithelial barrier permeability, remodelling and fibrosis) are attenuated in the lungs of Vim−/− mice challenged with LPS, bleomycin and asbestos. Bone marrow chimeric mice lacking vimentin have reduced IL-1β levels and attenuated lung injury and fibrosis following bleomycin exposure. Furthermore, decreased active caspase-1 and IL-1β levels are observed in vitro in Vim−/− and vimentin-knockdown macrophages. Importantly, we show direct protein–protein interaction between NLRP3 and vimentin. This study provides insights into lung inflammation and fibrosis and suggests that vimentin may be a key regulator of the NLRP3 inflammasome. The mechanism of NLRP3 activation remains incompletely characterized. Here the authors show that it is dependent on vimentin, and that NLRP3-mediated lung injury and fibrosis induced by endotoxin, asbestos or bleomycin are reduced in vimentin-deficient mice.

There is an accumulation of evidence in the literature demonstrating the integral role of vimentin intermediate filaments (IFs) in the progression of lung cancers. Vimentin IF proteins have been implicated in many aspects of cancer initiation and progression, including tumorigenesis, epithelial-to-mesenchymal transition (EMT), and the metastatic spread of cancer. Specifically, vimentin IFs have been recognized as an essential component regulating EMT, major signal transduction pathways involved in EMT and tumor progression, cell migration and invasion, the positioning and anchorage of organelles, such as mitochondria, and cell-cell and cell-substrate adhesion. In tumorgenesis, vimentin forms a complex with 14-3-3 and beclin 1 to inhibit autophagy via an AKT-dependent mechanism. Vimentin is a canonical marker of EMT, and recent evidence has shown it to be an important regulator of cellular motility. Transcriptional regulation of vimentin through hypoxia-inducible factor-1 may be a potential driver of EMT. Finally, vimentin regulates 14-3-3 complexes and controls various intracellular signaling and cell cycle control pathways by depleting the availability of free 14-3-3. There are many exciting advances in our understanding of the complex role of vimentin IFs in cancer, pointing to the key role vimentin IFs may play in tumor progression.

You have accessJournal of UrologyProstate Cancer: Basic Research (IV)1 Apr 2013797 TRANSCELLULAR MIGRATION OF PROSTATE CANCER CELLS THROUGH ENDOTHELIAL CELLS REQUIRES VIMENTIN Margo Quinn, Kristian Novakovic, Dale Shumaker, Sarah Rabbitt, Jaclyn Pruitt, and Brian Helfand Margo QuinnMargo Quinn Evanston, IL More articles by this author , Kristian NovakovicKristian Novakovic Evanston, IL More articles by this author , Dale ShumakerDale Shumaker Chicago, IL More articles by this author , Sarah RabbittSarah Rabbitt Evanston, IL More articles by this author , Jaclyn PruittJaclyn Pruitt Evanston, IL More articles by this author , and Brian HelfandBrian Helfand Evanston, IL More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.361AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Metastases to distant organs is a critical step in cancer progression and a major cause of mortality. To escape from the primary tumor, metastatic cancer cells must acquire the ability to migrate. This process, known as the epithelial to mesenchymal transition, often involves the expression of vimentin intermediate filaments (VIF). While it has previously been suggested that VIF are directly involved in cellular motility, it is unknown whether VIF are actively involved in prostate cancer (PC) invasion and metastases. METHODS Immunofluorescence and western blot analyses were used to compare VIF levels in several prostate cell lines: BPH-1, LNCaP, LNCap-81, PC3 cells, and subclones PC3-m, PC3-pro, PC3-LN4. VIF expression and assembly was inhibited using either vimentin siRNA or withaferin A (WFA,0.5-1μM, a natural inhibitor of VIF), and matrigel invasion assays were performed. PC3 cells expressing either Emerald-vimentin or Emerald-vimentin siRNA were placed on a monolayer of labeled CPAE endothelial cells and PC3 invasion through the CPAE cells was analyzed. The type of invasion (transcellular vs paracellular) was documented as well as the length of the invasion processes. Tumor growth was measured in a PC3 xenograft nude mouse model after intraperitoneal injection of either DMSO (control) or WFA (2.0mg/kg). RESULTS VIF expression correlated with the invasive capabilities of the prostate cell lines (PC3>LNCaP-81>LNCaP> BPH-1). Invasion was increased in LNCaP cells containing a vimentin construct vs a control vector (31% vs 8%; p<0.001). Cell invasion was significantly decreased in vimentin siRNA expressing LNCaP-81 cells compared to scramble-siRNA containing cells (1.8% vs 16%; p<0.001). More than 40% of PC3 cells exhibited transcellular invasion, with the remainder invading through the CPAE cell monolayer in a paracellular fashion. Live cell imaging showed that PC3 cells extend a long VIF-containing process (invasion process), necessary for trancellular invasion. VIF inhibition correlated with a significant (p<0.001) decrease in invasion process length, decrease in transcellular invasion (2%) and increased paracellular invasion (98%). Inhibition of VIF expression with WFA in PC-3 xenografts resulted in >65% reduction in tumor growth compared to controls. CONCLUSIONS Our results show that VIF are required for invasion process formation and PC transcellular invasion. Future investigation of VIF as a potential therapeutic target in metastatic PC should be considered. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e328 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Margo Quinn Evanston, IL More articles by this author Kristian Novakovic Evanston, IL More articles by this author Dale Shumaker Chicago, IL More articles by this author Sarah Rabbitt Evanston, IL More articles by this author Jaclyn Pruitt Evanston, IL More articles by this author Brian Helfand Evanston, IL More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Nuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved in the regulation of many nuclear functions. The results of this study demonstrate that LB1 expression in WI-38 cells decreases during cellular senescence. Premature senescence induced by oncogenic Ras also decreases LB1 expression through a retinoblastoma protein (pRb)-dependent mechanism. Silencing the expression of LB1 slows cell proliferation and induces premature senescence in WI-38 cells. The effects of LB1 silencing on proliferation require the activation of p53, but not pRb. However, the induction of premature senescence requires both p53 and pRb. The proliferation defects induced by silencing LB1 are accompanied by a p53-dependent reduction in mitochondrial reactive oxygen species (ROS), which can be rescued by growth under hypoxic conditions. In contrast to the effects of LB1 silencing, overexpression of LB1 increases the proliferation rate and delays the onset of senescence of WI-38 cells. This overexpression eventually leads to cell cycle arrest at the G1/S boundary. These results demonstrate the importance of LB1 in regulating the proliferation and senescence of human diploid cells through a ROS signaling pathway.

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011619 EPIGENETIC CONTROL OF ANDROGEN ENRICHED LAMIN DEFINED MICRODOMAINS (LDMDS) IN PROSTATE CANCER Brian T. Helfand, Yuanyuan Wang, Pekka Taiman, Katrin Pfleghaar, Robert D. Goldman, and Dale K. Shumaker Brian T. HelfandBrian T. Helfand Chicago, IL More articles by this author , Yuanyuan WangYuanyuan Wang Chicago, IL More articles by this author , Pekka TaimanPekka Taiman Chicago, IL More articles by this author , Katrin PfleghaarKatrin Pfleghaar Munich, Germany More articles by this author , Robert D. GoldmanRobert D. Goldman Chicago, IL More articles by this author , and Dale K. ShumakerDale K. Shumaker Chicago, IL More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1472AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Altered nuclear shape and DNA content have reproducibly been described in prostate cancer (CaP) specimens and are predictive of disease progression and metastasis. Nuclear lamin intermediate filaments are key determinants of nuclear size and shape and play a role in both DNA synthesis and transcription. We have shown that lamin-defined microdomains (LDMDs) are induced by knocking down lamin B1 (LB1) using shRNA. These LDMDs are enriched in euchromatin, accumulate activated RNA polymerase II (Pol IIo), are decreased in epigenetic histone marks for RNA elongation, and preferentially localize chromosome regions. The present study, 1) determined whether LDMDs are present within CaP cells and tissue and 2) characterized LDMD content. METHODS CaP tissue microarrays and human CaP cell lines with various invasive potential (DU-145, LNCaP, PC-3) were used. The presence and content of LDMDs were assayed using antibodies directed against lamin A/C, lamin B1, histone marks [AcH3 (euchromatin), H3K27me3 and H3K9me3 (heterochromatin), H3K36me3 (elongation phase of transcription)], Pol IIo and androgen receptor (AR). Transcription was assayed with BrUTP incorporation. RESULTS In CaP cells, LDMD presence appears to be correlated with disease aggressiveness and invasive potential. There was an increased frequency of LDMDs in high Gleason ≥8 disease vs. low grade (Gleason ≤6) disease (10.2% v. 2.1%; p<0.001). Based on immunostaining of epigenetic histone marks (AcH3, H3K27me3 and H3K9me3), LDMDs contain euchromatin which suggests a transcriptionally active microdomain. This is supported by the finding that LDMDs accumulate Pol IIo, and AR. Surprisingly, LDMDs are devoid of histone marks for the elongation phase of transcription and do not incorporate BrUTP. CONCLUSIONS LDMDs are present in CaP cells and their frequency appears to be correlated with tumor aggressiveness. LDMDs appear to be enriched for euchromatin. Although LDMDs accumulate AR and Pol IIo they appear to define a promoter proximal stalled state for Pol II. These studies will provide significant insights into the functions of LDMDs and will help to elucidate the mechanisms underlying the development and progression of aggressive CaP. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e249 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Brian T. Helfand Chicago, IL More articles by this author Yuanyuan Wang Chicago, IL More articles by this author Pekka Taiman Chicago, IL More articles by this author Katrin Pfleghaar Munich, Germany More articles by this author Robert D. Goldman Chicago, IL More articles by this author Dale K. Shumaker Chicago, IL More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

We report a gold nanoparticle-templated high density lipoprotein (HDL AuNP) platform for gene therapy that combines lipid-based nucleic acid transfection strategies with HDL biomimicry. For proof-of-concept, HDL AuNPs are shown to adsorb antisense cholesterylated DNA. The conjugates are internalized by human cells, can be tracked within cells using transmission electron microscopy, and regulate target gene expression. Overall, the ability to directly image the AuNP core within cells, the chemical tailorability of the HDL AuNP platform, and the potential for cell-specific targeting afforded by HDL biomimicry make this platform appealing for nucleic acid delivery.

Vimentin intermediate filaments (VIF) extend throughout the rear and perinuclear regions of migrating fibroblasts, but only nonfilamentous vimentin particles are present in lamellipodial regions. In contrast, VIF networks extend to the entire cell periphery in serum-starved or nonmotile fibroblasts. Upon serum addition or activation of Rac1, VIF are rapidly phosphorylated at Ser-38, a p21-activated kinase phosphorylation site. This phosphorylation of vimentin is coincident with VIF disassembly at and retraction from the cell surface where lamellipodia form. Furthermore, local induction of photoactivatable Rac1 or the microinjection of a vimentin mimetic peptide (2B2) disassemble VIF at sites where lamellipodia subsequently form. When vimentin organization is disrupted by a dominant-negative mutant or by silencing, there is a loss of polarity, as evidenced by the formation of lamellipodia encircling the entire cell, as well as reduced cell motility. These findings demonstrate an antagonistic relationship between VIF and the formation of lamellipodia.

The lamins are major determinants of nuclear shape and chromatin organization and these features are frequently altered in prostate cancer (CaP). Human CaP cell lines frequently have nuclear lobulations, which are enriched in A‐type lamins but lack B‐type lamins and have been defined as lamin B‐deficient microdomains (LDMDs). LDMD frequency is correlated with CaP cell line aggressiveness and increased cell motility. In addition, LNCaP cells grown in the presence of dihydrotestosterone (DHT) show an increased frequency of LDMDs. The LDMDs are enriched in activated RNA polymerase II (Pol IIo) and androgen receptor (AR) and A‐type lamins form an enlarged meshwork that appears to co‐align with chromatin fibres and AR. Furthermore, fluorescence in situ hybridization and comparative genomic hybridization demonstrated that chromosomal regions associated with CaP susceptibility are preferentially localized to LDMDs. Surprisingly, these regions lack histone marks for transcript elongation and exhibit reduced BrU incorporation, suggesting that Pol II is stalled within LDMDs. Real‐time PCR of genes near androgen response elements (AREs) was used to compare transcription between cells containing LDMDs and controls. Genes preferentially localized to LDMDs showed significantly decreased expression, while genes in the main nuclear body were largely unaffected. Furthermore, LDMDs were observed in human CaP tissue and the frequency was correlated with increased Gleason grade. These results imply that lamins are involved in chromatin organization and Pol II transcription, and provide insights into the development and progression of CaP. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The nuclear envelope (NE) is a double membrane physical barrier, which separates the nucleus from the cytoplasm. Underlying the NE are the nuclear lamins, which in combination with inner nuclear membrane proteins form the lamina. The lamina is crucial for maintaining the structural integrity of the nucleus and for positioning of nuclear pore complexes (NPCs) within the NE. The nucleoporin Nup153 has previously been reported to bind to B-type lamins. However, the specificity of this interaction is not well established. Here we show that Nup153 exhibits multiple binding sites for A- and B-type lamins. Using GST-pull down assays, we found that both the N-terminal domain of Nup153 and its C terminus associate with the Ig-fold domain of A- and B-type lamins. By employing purified Nup153 and lamin proteins in blot overlay assays we revealed that both the N-terminal and the C-terminal domain of Nup153 are directly interacting with the lamins. Moreover, we provide evidence that mutations in the lamin A Ig-fold domain selectively affect Nup153-binding, suggesting that Nup153 may play a role in lamin-associated diseases, known as laminopathies. Together our results indicate a far more intricate interplay between Nup153 and nuclear lamins than previously accepted.

The nucleoporin Nup153 is known to play pivotal roles in nuclear import and export in interphase cells and as the cell transitions into mitosis, Nup153 is involved in nuclear envelope breakdown. In this study, we demonstrate that the interaction of Nup153 with the spindle assembly checkpoint protein Mad1 is important in the regulation of the spindle checkpoint. Overexpression of human Nup153 in HeLa cells leads to the appearance of multinucleated cells and induces the formation of multipolar spindles. Importantly, it causes inactivation of the spindle checkpoint due to hypophosphorylation of Mad1. Depletion of Nup153 using RNA interference results in the decline of Mad1 at nuclear pores during interphase and more significantly causes a delayed dissociation of Mad1 from kinetochores in metaphase and an increase in the number of unresolved midbodies. In the absence of Nup153 the spindle checkpoint remains active. In vitro studies indicate direct binding of Mad1 to the N-terminal domain of Nup153. Importantly, Nup153 binding to Mad1 affects Mad1's phosphorylation status, but not its ability to interact with Mad2. Our data suggest that Nup153 levels regulate the localization of Mad1 during the metaphase/anaphase transition thereby affecting its phoshorylation status and in turn spindle checkpoint activity and mitotic exit.

Intermediate filament (IF) dynamics during organelle transport and their role in organelle movement were studied using Xenopus laevis melanophores. In these cells, pigment granules (melanosomes) move along microtubules and microfilaments, toward and away from the cell periphery in response to α-melanocyte stimulating hormone (α-MSH) and melatonin, respectively. In this study we show that melanophores possess a complex network of vimentin IFs which interact with melanosomes. IFs form an intricate, honeycomb-like network that form cages surrounding individual and small clusters of melanosomes, both when they are aggregated and dispersed. Purified melanosome preparations contain a substantial amount of vimentin, suggesting that melanosomes bind to IFs. Analyses of individual melanosome movements in cells with disrupted IF networks show increased movement of granules in both anterograde and retrograde directions, further supporting the notion of a melanosome-IF interaction. Live imaging reveals that IFs, in turn, become highly flexible as melanosomes disperse in response to α-MSH. During the height of dispersion there is a marked increase in the rate of fluorescence recovery after photobleaching of GFP-vimentin IFs and an increase in vimentin solubility. These results reveal a dynamic interaction between membrane bound pigment granules and IFs and suggest a role for IFs as modulators of granule movement.

Over the past few years it has become evident that the intermediate filament proteins, the types A and B nuclear lamins, not only provide a structural framework for the nucleus, but are also essential for many aspects of normal nuclear function. Insights into lamin-related functions have been derived from studies of the remarkably large number of disease-causing mutations in the human lamin A gene. This review provides an up-to-date overview of the functions of nuclear lamins, emphasizing their roles in epigenetics, chromatin organization, DNA replication, transcription, and DNA repair. In addition, we discuss recent evidence supporting the importance of lamins in viral infections.

The nuclear lamins function in the regulation of replication, transcription, and epigenetic modifications of chromatin. However, the mechanisms responsible for these lamin functions are poorly understood. We demonstrate that A- and B-type lamins form separate, but interacting, stable meshworks in the lamina and have different mobilities in the nucleoplasm as determined by fluorescence correlation spectroscopy (FCS). Silencing lamin B1 (LB1) expression dramatically increases the lamina meshwork size and the mobility of nucleoplasmic lamin A (LA). The changes in lamina mesh size are coupled to the formation of LA/C-rich nuclear envelope blebs deficient in LB2. Comparative genomic hybridization (CGH) analyses of microdissected blebs, fluorescence in situ hybridization (FISH), and immunofluorescence localization of modified histones demonstrate that gene-rich euchromatin associates with the LA/C blebs. Enrichment of hyperphosphorylated RNA polymerase II (Pol II) and histone marks for active transcription suggest that blebs are transcriptionally active. However, in vivo labeling of RNA indicates that transcription is decreased, suggesting that the LA/C-rich microenvironment induces promoter proximal stalling of Pol II. We propose that different lamins are organized into separate, but interacting, microdomains and that LB1 is essential for their organization. Our evidence suggests that the organization and regulation of chromatin are influenced by interconnections between these lamin microdomains.

This study provides insights into the role of nuclear lamins in DNA replication. Our data demonstrate that the Ig-fold motif located in the lamin C terminus binds directly to proliferating cell nuclear antigen (PCNA), the processivity factor necessary for the chain elongation phase of DNA replication. We find that the introduction of a mutation in the Ig-fold, which alters its structure and causes human muscular dystrophy, inhibits PCNA binding. Studies of nuclear assembly and DNA replication show that lamins, PCNA, and chromatin are closely associated in situ. Exposure of replicating nuclei to an excess of the lamin domain containing the Ig-fold inhibits DNA replication in a concentration-dependent fashion. This inhibitory effect is significantly diminished in nuclei exposed to the same domain bearing the Ig-fold mutation. Using the crystal structures of the lamin Ig-fold and PCNA, molecular docking simulations suggest probable interaction sites. These findings also provide insights into the mechanisms underlying the numerous disease-causing mutations located within the lamin Ig-fold.

Mitotic spindle morphogenesis is a series of highly coordinated movements that lead to chromosome segregation and cytokinesis. We report that the intermediate filament protein lamin B, a component of the interphase nuclear lamina, functions in spindle assembly. Lamin B assembled into a matrix-like network in mitosis through a process that depended on the presence of the guanosine triphosphate-bound form of the small guanosine triphosphatase Ran. Depletion of lamin B resulted in defects in spindle assembly. Dominant negative mutant lamin B proteins that disrupt lamin B assembly in interphase nuclei also disrupted spindle assembly in mitosis. Furthermore, lamin B was essential for the formation of the mitotic matrix that tethers a number of spindle assembly factors. We propose that lamin B is a structural component of the long-sought-after spindle matrix that promotes microtubule assembly and organization in mitosis.