Vimentin intermediate filaments and filamentous actin form unexpected interpenetrating networks that redefine the cell cortex

The cytoskeleton of eukaryotic cells is primarily composed of networks of filamentous proteins, F-actin, microtubules, and intermediate filaments. Interactions among the cytoskeletal components are important in determining cell structure and in regulating cell functions. For example, F-actin and microtubules work together to control cell shape and polarity, while the subcellular organization and transport of vimentin intermediate filament (VIF) networks depend on their interactions with microtubules. However, it is generally thought that F-actin and VIFs form two coexisting but separate networks that are independent due to observed differences in their spatial distribution and functions. In this paper, we present a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks. We characterize the structure of VIFs and F-actin networks within the cell cortex using structured illumination microscopy and cryo-electron tomography. We find that VIFs and F-actin form an interpenetrating network (IPN) with interactions at multiple length scales, and VIFs are integral components of F-actin stress fibers. From measurements of recovery of cell contractility after transient stretching, we find that the IPN structure results in enhanced contractile forces and contributes to cell resilience. Studies of reconstituted networks and dynamic measurements in cells suggest direct and specific associations between VIFs and F-actin. From these results, we conclude that VIFs and F-actin work synergistically, both in their structure and in their function. These results profoundly alter our understanding of the contributions of the components of the cytoskeleton, particularly the interactions between intermediate filaments and F-actin.

The cytoskeleton is a highly dynamic structure composed of multiple types of filamentous proteins. In eukaryotic cells, actin, microtubules, and intermediate filaments (IFs) each form intricate networks of entangled and cross-linked filaments. The organization of each individual network is precisely controlled to enable essential cellular functions. However, many core processes also require interactions among the different cytoskeletal components. For example, filamentous-actin (F-actin) and microtubules work together to control cell shape and polarity, which are critical for development, cell migration, and division. Close associations between microtubules and vimentin IFs (VIFs) have also been proposed based on similarities in their spatial distributions and the dependence of the organization of VIF networks on the microtubule-associated motors, kinesin and dynein (1–3). Indeed, there is some experimental evidence that microtubules can template VIF assembly and that VIFs can guide microtubules (4, 5), while VIFs stabilize microtubules in vitro (6). In addition, in stratified epithelial cells, a subplasmalemmal rim of keratin IFs can be localized just below the actin cortex, suggesting cooperativity of keratin and actin networks in regulating cell mechanics (7). Despite such interactions, VIFs and F-actin are generally thought to form two coexisting but separate networks. For example, fluorescence microscopy typically reveals the strongest signals for F-actin in the cell periphery, whereas the strongest signals for VIFs are near the nucleus in the bulk cytoplasm, suggesting that the two networks have little or no interaction. Furthermore, the functions of F-actin and VIFs appear to be largely contrasting: F-actin generates forces, whereas VIFs provide stability against these forces. Nevertheless, some evidence suggests there may be connections between vimentin and actin: for example, vimentin knockout cells are less motile and less contractile than their wild-type (WT) counterparts (8). Furthermore, some interactions have been observed between F-actin and VIFs (9–11) as well as the precursors to keratin, another IF system (12). These findings suggest that direct interactions or connections may exist between VIFs and F-actin. However, there have been no reports of direct observations of these interactions through imaging or other means, which would provide conclusive evidence of their significance. Such connections would belie our current understanding of the two independent cytoskeletal networks but could have a profound effect on the mechanical properties of cells. The possibility of such connections demands a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks.Here we present evidence that VIFs and F-actin do work synergistically and form an interpenetrating network (IPN) structure within the cell cortex, defined as the cortical cytoplasm adjacent to the cell surface. We combine high-resolution structured illumination microscopy (SIM) and cryo-electron tomography (cryo-ET) to image mouse embryonic fibroblasts (MEFs) and observe coupling between F-actin and VIF structures within the cortex, contrary to the widely accepted view that they are each spatially segregated. In fact, the association of VIFs with cortical arrays of F-actin stress fibers occurs at multiple length scales. For example, VIFs run through and frequently appear to interconnect with adjacent stress fibers, forming meshworks that surround them. These organizational states are consistent with the formation of an IPN. We show that this IPN structure has important functional consequences in cells and can result in enhanced contractile forces. Moreover, our results indicate that specific associations exist between actin and vimentin proteins in the cytoplasmic environment, which may facilitate the formation of an IPN; the results also show that the VIF network can influence the diffusive behavior of actin monomers, which may, in turn, have downstream effects on other actin-driven processes. Thus, vimentin has a far more comprehensive role in cellular function than previously thought. These findings confirm the importance of the interplay between VIFs and F-actin, especially as it relates to the formation of IPNs and their consequences on the contractile nature of cells.     

Characterization of Hantavirus N Protein Intracellular Dynamics and Localization

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation, and several other crucial processes during hantavirus infection. In this study, we generated fluorescently tagged N protein constructs derived from Puumalavirus (PUUV), the dominant hantavirus species in Central, Northern, and Eastern Europe. We comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We observed formation of large, fibrillar PUUV N protein aggregates, rapidly coalescing from early punctate and spike-like assemblies. Moreover, we found significant spatial correlation of N with vimentin, actin, and P-bodies but not with microtubules. N constructs also co-localized with Gn and Gc albeit not as strongly as the glycoproteins associated with each other. Finally, we assessed oligomerization of N constructs, observing efficient and concentration-dependent multimerization, with complexes comprising more than 10 individual proteins.     

Abnormal distribution of desmin and vimentin in myofibers in adult onset myotubular myopathy.

We report, for the first time, muscle immunocytochemical studies in sporadic, adult onset myotubular myopathy (SAOMM), which show intramyofibrillar central, perinuclear desmin and vimentin. This pattern was absent in a normal control and in myofibers with increased internal nuclei associated with denervation and myotonic muscular dystrophy (MyD). These findings resemble those reported in 8- to 15-week-old human fetal myotubes and myofibers of infantile MM, implying a possible regression of intermediate filaments of adult myofibers to an early developmental phase in SAOMM.     

Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis: As Good as it Gets?

Anti-citrullinated protein antibodies (ACPAs) have recently emerged as sensitive and specific serological markers of rheumatoid arthritis (RA), providing superior alternative of the rheumatoid factor (RF) test in the laboratory diagnostics of RA. The first members of this autoantibody family were anti-perinuclear factor (APF) and anti-keratin antibodies (AKA). It became evident that both APF and AKA recognize citrullinated epitopes of filaggrin. Citrullination is a post-translational modification of arginine by deimination, physiologically occurring during apoptosis, inflammation or keratinization. The presence of several citrullinated proteins has been demonstrated in the RA synovium. The identification of citrullinated epitopes as targets for anti-filaggrin antibodies led to the development of the first and later second generation anti-cyclic citrullinated peptide (anti-CCP) antibody assays. The widely used anti-CCP2 assays have high diagnostic sensitivity and specificity, and they also show important predictive and prognostic value in RA. The anti-Sa antibody has been identified a decade ago; however, recent studies confirmed that anti-Sa is directed against citrullinated vimentin, hence it is a new member of the family of ACPAs. The newly developed anti-mutated citrullinated vimentin (anti-MCV) assay has similar diagnostic performance than the anti-CCP2 ELISA; however, the diagnostic spectrum of the anti-MCV test is somewhat different from that of anti-CCP2. It’s especially useful in the diagnosis of RA in RF and anti-CCP2 seronegative patients. The combined application of anti-CCP2 and anti-MCV assays can improve the laboratory diagnostics of RA. The family of ACPAs is expected to expand; there is an increasing need for developing new diagnostic strategies after careful evaluation of the characteristics of the available assays. Zoltán Szekanecz and Lilla Soós with equal contribution.     

Human decidual stromal cells express alpha-smooth muscle actin and show ultrastructural similarities with myofibroblasts.

Previous reports in human and mouse material demonstrated that decidual stromal cells expressed antigens associated with haematopoietic cells, exerted immune functions, and originated from bone marrow. These findings suggested that these cells belonged to the haematopoietic lineage. We purified and expanded in culture precursors of human decidual stromal cells, and found in electron microscopic images that the ultrastructure of these cells was similar to that of myofibroblasts, which are of mesenchymal origin. The relationship between these two types of cell was confirmed by the detection (by flow cytometry) in the decidual precursors of alpha-smooth muscle actin, a contractile microfilament expressed solely by smooth muscle cells, myofibroblasts and related cells. This filament was also detected in decidual stromal cells decidualized in vitro by the effect of progesterone. We also found vimentin in decidual precursors and decidualized cells. This intermediate filament has been previously reported to be expressed by all decidual stromal cells and also by myofibroblasts. Desmin, another intermediate filament expressed by myofibroblasts, was not detected in the decidual precursors; however, this filament was observed in decidualized cells. The expression of alpha-smooth muscle actin by decidual stromal cells was also found by immunostaining in cryostat sections of early decidua. Our results suggest that decidual stromal cells are related to myofibroblasts.     

The cytokeratin profile of medullary carcinoma of the breast

AIMS: The cytokeratin (CK) phenotype and vimentin expression of 31 medullary carcinomas was studied using commercially available antibodies on archived material. Comparing the phenotype of typical and atypical tumours and the phenotype of metastases, the biological significance of cytokeratin and vimentin expression in medullary carcinomas of the breast was determined. METHODS AND RESULTS: Antibodies to CK4, CK5 and 6, CK7, CK14, CK8 and 18, CK19, CK20 and to vimentin were used. All the typical and atypical medullary carcinomas and the metastases (10 cases) stained negatively for CK4 and positively for CK8-18 (CAM5.2). Almost all the tumours were CK7 and CK19 positive and CK20 negative. Twelve per cent of the tumours contained CK14. Twenty-five per cent of the typical, 43% of the atypical and 20% of the metastatic medullary carcinomas showed CK5-6 positivity. No association between the cytokeratin-vimentin profile of the tumours and axillary node metastases, tumour size or oestrogen receptor status was found but instability of CK expression was demonstrated by comparing the primary tumours with their metastases. CONCLUSIONS: : Medullary carcinomas of the breast express all the glandular type CKs including CK19 and additionally a proportion of the tumours expresses some of the CKs typical for myoepithelial cells. There was no correlation with prognostic factors.     

波形蛋白与DNA拓扑异构酶Ⅱ在病理性瘢痕中的表达及意义

目的:比较波形蛋白及DNA拓扑异构酶Ⅱα(TopoⅡα)、β(TopoⅡβ)在病理性瘢痕、成熟瘢痕和正常皮肤中的表达情况,探讨它们与成纤维细胞生物学特征的关系及在瘢痕形成中的作用.方法:应用免疫组织化学检测病理性瘢痕、成熟瘢痕和正常皮肤各10例,了解其中波形蛋白、TopoⅡα、β的表达水平,测定光密度值,并分析其相关性.结果:波形蛋白在病理性瘢痕和成熟瘢痕真皮层大量表达;TopoⅡα蛋白在病理性瘢痕表皮基底层表达明显低于正常皮肤和成熟瘢痕,但是在真皮层血管周围有阳性表达;TopoⅡβ蛋白在病理性瘢痕表皮层表达明显低于正常皮肤和成熟瘢痕,但是在真皮层胶原纤维增生区域大量表达.各组光密度值差异有统计学意义.结论:TopoⅡα、β在病理性瘢痕真皮层表达增强,与波形蛋白的表达一致,提示其可能与病理性瘢痕的形成有关.     

白细胞介素8对宫颈癌细胞增殖和侵袭转移的影响及其机制*

目的：探讨白细胞介素8（ IL-8） 对宫颈癌细胞Caski 增殖、侵袭转移的影响及其机制。方法：将 体外培养的宫颈癌Caski细胞分为对照组、IL-8 组（20、40、60、80、100 ng/mL）、IL-8+LY294002 组 （80 ng/mL IL-8+20 μmol/L LY294002）以及LY294002 组（20 μmol/L）,采用MTT 实验检测Caski 细胞的增殖能力, 采用划痕实验检测Caski 细胞的侵袭能力,采用Transwell 实验检测Caski 细胞的迁移能力。免疫印迹法检测蛋白 激酶B（ PKB）、磷酸化蛋白激酶B（ p-AKT）、波形蛋白、β-连环蛋白、上皮-钙黏蛋白（ E-cadherin）的表达情 况。结果： IL-8 呈剂量依赖性促进细胞增殖,80 ng/mL 与100 ng/mL 组之间差异无统计学意义。IL-8 组划痕愈合 能力和细胞迁移能力均增强,IL-8+LY294002 组及LY294002 组划痕愈合能力和细胞迁移能力均低于IL-8 组。各 组AKT总蛋白表达量差异无统计学意义,IL-8 组中p-AKT、波形蛋白和β- 连环蛋白表达均增加,E-cadherin 减少, AKT通路特异性抑制剂LY294002 可阻断上述作用。结论：IL-8 可能通过PI3K/AKT 通路影响波形蛋白、β- 连环 蛋白和E-cadherin 蛋白的表达,促进宫颈癌细胞的增殖、侵袭和转移。     

Expression of c-ets-1 mRNA is associated with an invasive, EMT-derived phenotype in breast carcinoma cell lines

We have previously observed in vitro that some stromal proteinases (MMP-2, MT1-MMP) were expressed or activated by invasive carcinoma cell lines exhibiting mesenchymal features, presumably acquired through an epithelial to mesenchymal transition (EMT). To examine the potential contribution of c-ets-1 to this phenotype, we have compared here the expression of c-ets-1 with invasiveness in vitro and expression of vimentin, E-cadherin, uPA, MMP-1 and MMP-3 in a panel of human breast cancer cell lines. Our results clearly demonstrate an association between c-ets-1 expression and the invasive, EMT-derived phenotype, which is typified by the expression of vimentin and the lack of E-cadherin. While absent from the two non-invasive, vimentin-negative cell lines, c-ets-1 was abundantly expressed in all the four vimentin-positive lines. However, we could not find a clear quantitative or qualitative relationship between the expression of c-ets-1 and the three proteinases known to be regulated by c-ets-1, except that when they were expressed, it was only in the invasive c-ets-1-positive lines. UPA mRNAs were found in three of the four vimentin-positive lines, MMP-1 in two of the four, and MMP-3 could not be detected in any of the cell lines. Intriguingly, MDA-MB-435 cells, which exhibit the highest metastatic potential of these cell lines in nude mice, expressed vimentin and c-ets-1, but lacked expression of these three proteinases, at least under the culture conditions employed. Taken together, our results show that c-ets-1 expression is associated with an invasive, EMT-derived phenotype in breast cancer cells, although it is apparently not sufficient to ensure the expression of uPA, MMP-1 or MMP-3, in the vimentin-positive cells. Such proteases regulation is undoubtedly qualified by the cellular context. This study therefore advances our understanding of the molecular regulation of invasiveness in EMT-associated carcinoma progression, and suggests that c-ets-1 may contribute to the invasive phenotype in carcinoma cells.     

Human intervertebral disc cell morphology and cytoskeletal composition: a preliminary study of regional variations in health and disease

Classically, intervertebral disc cells have been described as fibrocytic in the anulus fibrosus and chondrocytic in the nucleus pulposus. Recent animal studies, however, have suggested that disc cell morphology may be more complex than previously considered. Here, by utilizing labelling of components of the cytoskeleton in combination with confocal microscopy, we have examined the detailed morphology of human intervertebral disc cells in pathological and non-pathological tissue. Filamentous-actin- and vimentin-positive cells that appeared either fibrocytic or chondrocytic were observed in all intervertebral discs. However, in localized areas of the disc, stellate cells that extended multiple, branching cytoplasmic processes into their surrounding matrix were also seen. This stellate appearance formed a marked feature of disc cells regionally in certain pathologies, i.e. in cells of the outer anulus fibrosus in scoliotic discs and in inner anulus/nucleus pulposus cells in one spondylolisthetic disc. We conclude that the phenotypic variation of human intervertebral disc cells should be extended to include cells with a stellate appearance, which may be more prevalent in tissue that has been subjected to abnormal load or tension.