Non-mechanical Functions Research Articles

Mechanical and Non-Mechanical Functions of Filamentous and Non-Filamentous Vimentin.

Intermediate filaments (IFs) formed by vimentin are less understood than their cytoskeletal partners, microtubules and F-actin, but the unique physical properties of IFs, especially their resistance to large deformations, initially suggest a mechanical function. Indeed, vimentin IFs help regulate cell mechanics and contractility, and in crowded 3D environments they protect the nucleus during cell migration. Recently, a multitude of studies, often using genetic or proteomic screenings show that vimentin has many non-mechanical functions within and outside of cells. These include signaling roles in wound healing, lipogenesis, sterol processing, and various functions related to extracellular and cell surface vimentin. Extracellular vimentin is implicated in marking circulating tumor cells, promoting neural repair, and mediating the invasion of host cells by viruses, including SARS-CoV, or bacteria such as Listeria and Streptococcus. These findings underscore the fundamental role of vimentin in not only cell mechanics but also a range of physiological functions. Also see the video abstract here https://youtu.be/YPfoddqvz-g.

Abstract 3063: Stem cell lineage hierarchy by keratin profiling in normal human prostate epithelial cells and prostate cancer

Abstract Background: Both mechanical and nonmechanical functions of keratins have been implicated in stem cells and cancers. Using a sphere-based label retention assay, we recently isolated and characterized prostate stem cells and cancer stem-like cells from mixed progenitor populations (PMID 28651114), identifying keratin 13 (KRT13) as a specific prostate stem cell marker regulating self-renewal. Herein we utilize detailed keratin profiles to further clarify the human prostate epithelial lineage hierarchy and identify prostate cancer stem-like cells. Methods and Results: Primary prostate epithelial cells were 3D cultured (5 days) to form prostaspheres (PS), followed by PS-based long-term label retention and FACS to separate stem cells from progenitors. In normal prostate tissues from three healthy donors, RNA-seq revealed enrichment of KRT13, 23, 80, 78, 86 and 4 in label-retaining prostate stem cells while KRT6, 17, 14, 5, 8, 18 and P63 were enriched in nonretaining progenitors. We next used Fluidigm C1 captured single-cell RNA-seq and identified three major clusters in the label-retaining stem cell population; Cluster I represents quiescent stem cells (KRT13, 23, 80, 78, 4 enriched) and Clusters II and III contain active stem cells and bipotent progenitors, respectively (KRT16, 17, 6 enriched). GSEA analysis found stem cell and cancer-related pathways enrichment in Cluster I. Three additional clusters were identified in nonretaining progenitor cells, with Cluster IV representing unipotent basal progenitor cells (KRT5, 14, 6, 16 enriched) and Clusters V and VI early- and late-stage unipotent luminal progenitors (KRT8, 18, 10 enriched). Cancer stem-like cells were similarly isolated from three prostate cancer specimens and RNA-seq with MetaCore pathway analysis found enrichment of cytoskeleton remodeling keratin filaments. Interestingly, in addition to normal stem cell keratins (KRT13, 23, 80, 78, 4), other keratins (KRT10, 19, 6, 75, 16, 79, 3, 82) were enriched in cancer stem-like cells. Surprisingly, stem-like cells from patient-matched benign regions revealed a similar keratin profile, suggesting a cancer field effect for stem-like cell populations. Conclusion: Taken together, using gene profiling with an emphasis on keratin patterns, we have delineated the lineage hierarchy of human prostate stem cells originating from the activation of quiescent stem cells to bipotent progenitors that give rise to unipotent basal and luminal progenitor cells. We have identified common keratins enriched in stem cells from normal prostate and cancer/benign tissues as well as keratins unique in stem-like cells from prostate cancer. This clarification of the stem cell lineage hierarchy and keratin profiling of human prostate stem cells and cancer stem-like cells may provide enhanced opportunities for translational studies that target therapeutic-resistant cancer stem-like cells. (CA-172220) Citation Format: Wenyang Hu, Danping Hu, Lishi Xie, Hong Hu, Ye Li, Larisa Nonn, Toshi Shioda, Gail S. Prins. Stem cell lineage hierarchy by keratin profiling in normal human prostate epithelial cells and prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3063.

Types I and II Keratin Intermediate Filaments.

Keratins-types I and II-are the intermediate-filament-forming proteins expressed in epithelial cells. They are encoded by 54 evolutionarily conserved genes (28 type I, 26 type II) and regulated in a pairwise and tissue type-, differentiation-, and context-dependent manner. Here, we review how keratins serve multiple homeostatic and stress-triggered mechanical and nonmechanical functions, including maintenance of cellular integrity, regulation of cell growth and migration, and protection from apoptosis. These functions are tightly regulated by posttranslational modifications and keratin-associated proteins. Genetically determined alterations in keratin-coding sequences underlie highly penetrant and rare disorders whose pathophysiology reflects cell fragility or altered tissue homeostasis. Furthermore, keratin mutation or misregulation represents risk factors or genetic modifiers for several additional acute and chronic diseases.

Influence of body weight on bone mass, architecture and turnover.

Weight-dependent loading of the skeleton plays an important role in establishing and maintaining bone mass and strength. This review focuses on mechanical signaling induced by body weight as an essential mechanism for maintaining bone health. In addition, the skeletal effects of deviation from normal weight are discussed. The magnitude of mechanical strain experienced by bone during normal activities is remarkably similar among vertebrates, regardless of size, supporting the existence of a conserved regulatory mechanism, or mechanostat, that senses mechanical strain. The mechanostat functions as an adaptive mechanism to optimize bone mass and architecture based on prevailing mechanical strain. Changes in weight, due to altered mass, weightlessness (spaceflight), and hypergravity (modeled by centrifugation), induce an adaptive skeletal response. However, the precise mechanisms governing the skeletal response are incompletely understood. Furthermore, establishing whether the adaptive response maintains the mechanical competence of the skeleton has proven difficult, necessitating the development of surrogate measures of bone quality. The mechanostat is influenced by regulatory inputs to facilitate non-mechanical functions of the skeleton, such as mineral homeostasis, as well as hormones and energy/nutrient availability that support bone metabolism. Although the skeleton is very capable of adapting to changes in weight, the mechanostat has limits. At the limits, extreme deviations from normal weight and body composition are associated with impaired optimization of bone strength to prevailing body size.

Functional Analysis of Keratin-Associated Proteins in Intestinal Epithelia: Heat-Shock Protein Chaperoning and Kinase Rescue.

A growing body of evidence from several laboratories points at nonmechanical functions of keratin intermediate filaments (IF), such as control of apoptosis, modulation of signaling, or regulation of innate immunity, among others. While these functions are generally assigned to the ability of IF to scaffold other proteins, direct mechanistic causal relationships between filamentous keratins and the observed effects of keratin knockout or mutations are still missing. We have proposed that the scaffolding of chaperones such as Hsp70/40 may be key to understand some IF nonmechanical functions if unique features or specificity of the chaperoning activity in the IF scaffold can be demonstrated. The same criteria of uniqueness could be applied to other biochemical functions of the IF scaffold. Here, we describe a subcellular fractionation technique based on established methods of keratin purification. The resulting keratin-enriched fraction contains several proteins tightly associated with the IF scaffold, including Hsp70/40 chaperones. Being nondenaturing, this fractionation method enables direct testing of chaperoning and other enzymatic activities associated with IF, as well as supplementation experiments to determine the need for soluble (cytosolic) proteins. This method also permits to analyze inhibitory activity of cytosolic proteins at independently characterized physiological concentrations. When used as complementary approaches to knockout, knockdown, or site-directed mutagenesis, these techniques are expected to shed light on molecular mechanisms involved in the effects of IF loss of function.

Skin Keratins.

Keratins comprise the type I and type II intermediate filament-forming proteins and occur primarily in epithelial cells. They are encoded by 54 evolutionarily conserved genes (28 type I, 26 type II) and regulated in a pairwise and tissue type-, differentiation-, and context-dependent manner. Keratins serve multiple homeostatic and stress-enhanced mechanical and nonmechanical functions in epithelia, including the maintenance of cellular integrity, regulation of cell growth and migration, and protection from apoptosis. These functions are tightly regulated by posttranslational modifications as well as keratin-associated proteins. Genetically determined alterations in keratin-coding sequences underlie highly penetrant and rare disorders whose pathophysiology reflects cell fragility and/or altered tissue homeostasis. Moreover, keratin mutation or misregulation represents risk factors or genetic modifiers for several acute and chronic diseases. This chapter focuses on keratins that are expressed in skin epithelia, and details a number of basic protocols and assays that have proven useful for analyses being carried out in skin.

Abstract 1958: Interaction between keratin intermediate filament proteins K8/18 and cancer related signal transduction proteins in epithelial cells

Abstract Intermediate filaments (IFs) are cytoskeletal polymers formed by a large family of proteins that are expressed in a cell differentiation and tissue specific manner. Keratins (K) are the IF proteins present in epithelial cells. They are known to provide structural supports to cells and play roles in maintaining cellular integrity against mechanical and toxic stresses. More recently, keratins have been recognized to play non-mechanical functions which include the regulation of signaling pathways that control cell growth and survival. In tumor pathology, K8 and K18 which are the only keratin present in hepatocytes were, for a long time, just considered as cell type markers. However, in recent years various studies have provided evidence that keratins should be considered as regulators of cancer cell signaling. For instance, K8/18 loss is a hallmark of epithelial mesenchymal transition (EMT) but their role in tumor progression is unclear. In an attempt to bring more insight into the function of keratins in this process, we first investigated whether K8/18 expression played an active role in EMT. Using shRNA, we produced new K8/18 stable knockdown cell lines (human endometrial carcinoma KLE, human hepatocellular carcinoma HepG2 and human cervical carcinoma HeLa cell). Our K8/18 stable knockdown model presented a hyperactivation of the PI3K/Akt/NF-κB axis. Moreover, these changes were associated with an increase in collective cell migration and invasiveness. Because of these results, we considered whether NF-κB and its activators (PI3K, Akt (1, 2, 3), pAkt, IKK (α, β, γ), IκB-α and pIκB-α) are associated with keratins in epithelial cells. Using immunoprecipitation and immunofluorescence, our current results show that pAkt, NF-κB and IKKβ are indeed associated with keratins. Taken together, these results support the hypothesis that keratin intermediate filament proteins provide a platform for these proteins involved in signal transduction. Further studies will be done to determine if posttranslational modifications that affect keratins (K8/18 phosphorylation on specific serines and/or K18 O-glycosylation) interfere with their association to signaling protein and transcription factors previously found. Changes in K8/18 posttranlational modifications and their association with different proteins could be important determinants in cancer progression of epithelial cells. This work was supported by CRSNG grants to MC and EA. Citation Format: Stephanie Lamontagne, Anne-Marie Fortier, Sophie Parent, Eric Asselin, Monique Cadrin. Interaction between keratin intermediate filament proteins K8/18 and cancer related signal transduction proteins in epithelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1958. doi:10.1158/1538-7445.AM2015-1958

Mechanical and non-mechanical functions of Dystrophin can prevent cardiac abnormalities in Drosophila

Dystrophin-deficiency causes cardiomyopathies and shortens the life expectancy of Duchenne and Becker muscular dystrophy patients. Restoring Dystrophin expression in the heart by gene transfer is a promising avenue to explore as a therapy. Truncated Dystrophin gene constructs have been engineered and shown to alleviate dystrophic skeletal muscle disease, but their potential in preventing the development of cardiomyopathy is not fully understood. In the present study, we found that either the mechanical or the signaling functions of Dystrophin were able to reduce the dilated heart phenotype of Dystrophin mutants in a Drosophila model. Our data suggest that Dystrophin retains some function in fly cardiomyocytes in the absence of a predicted mechanical link to the cytoskeleton. Interestingly, cardiac-specific manipulation of nitric oxide synthase expression also modulates cardiac function, which can in part be reversed by loss of Dystrophin function, further implying a signaling role of Dystrophin in the heart. These findings suggest that the signaling functions of Dystrophin protein are able to ameliorate the dilated cardiomyopathy, and thus might help to improve heart muscle function in micro-Dystrophin-based gene therapy approaches.

Lessons from the endothelial junctional mechanosensory complex

Mechanotransduction plays a key role in both normal physiology and in diseases such as cancer, atherosclerosis and hypertension. Nowhere is this more evident than in the vascular system, where fluid shear stress from blood flow plays a critical role in shaping the blood vessels and in determining their function and dysfunction. Responses to flow are mediated in part by a complex of proteins comprised of PECAM-1, VE-cadherin and VEGFR2 at endothelial cell-cell junctions; all proteins that clearly have other, non-mechanical functions. We review recent progress toward understanding the functions and mechanisms of mechanotransduction by this complex and suggest some principles that may apply more broadly.

Composite materials, composite structures, composite systems

AbstractThe first part of the history of composites in aerospace emphasised materials with high specific strength and stiffness. This was followed by a quest for reliable manufacturing techniques that guaranteed sufficiently high fibre volume fractions in complex structural parts with reasonable cost. Further improvements are still possible leading, ultimately to an extension of the functionality of composite structures to non-mechanical functions. Reduction of material scatter and a more probability-based design approach, improved material properties, higher post buckling factors, improved crashworthiness concepts and improved NDI techniques are some of the evolutionary measures that could improve the performance of current composite structures. Modular design, increased co-curing, hybrid material structures, hybrid fabrication methods, innovative structural concepts and reduced development times are more revolutionary steps that could bring today’s solutions further. Manufacturing engineering is also important for achieving revolutionary change. Function integration such as embedded deicing, morphing,, and boundary-layer suction are among the next steps in weight and cost reduction, but now on the system level.

Expression of the dystrophin isoform Dp116 preserves functional muscle mass and extends lifespan without preventing dystrophy in severely dystrophic mice

Dp116 is a non-muscle isoform of dystrophin that assembles the dystrophin-glycoprotein complex (DGC), but lacks actin-binding domains. To examine the functional role of the DGC, we expressed the Dp116 transgene in mice lacking both dystrophin and utrophin (mdx:utrn(-/-)). Unexpectedly, expression of Dp116 prevented the most severe aspects of the mdx:utrn(-/-) phenotype. Dp116:mdx:utrn(-/-) transgenic mice had dramatic improvements in growth, mobility and lifespan compared with controls. This was associated with increased muscle mass and force generating capacity of limb muscles, although myofiber size and specific force were unchanged. Conversely, Dp116 had no effect on dystrophic injury as determined by muscle histopathology and serum creatine kinase levels. Dp116 also failed to restore normal fiber-type distribution or the post-synaptic architecture of the neuromuscular junction. These data demonstrate that the DGC is critical for growth and maintenance of muscle mass, a function that is independent of the ability to prevent dystrophic pathophysiology. Likewise, this is the first demonstration in skeletal muscle of a positive functional role for a dystrophin protein that lacks actin-binding domains. We conclude that both mechanical and non-mechanical functions of dystrophin are important for its role in skeletal muscle.

Introducing intermediate filaments: from discovery to disease

It took more than 100 years before it was established that the proteins that form intermediate filaments (IFs) comprise a unified protein family, the members of which are ubiquitous in virtually all differentiated cells and present both in the cytoplasm and in the nucleus. However, during the past 2 decades, knowledge regarding the functions of these structures has been expanding rapidly. Many disease-related roles of IFs have been revealed. In some cases, the molecular mechanisms underlying these diseases reflect disturbances in the functions traditionally assigned to IFs, i.e., maintenance of structural and mechanical integrity of cells and tissues. However, many disease conditions seem to link to the nonmechanical functions of IFs, many of which have been defined only in the past few years.

The role of the hepatocyte cytokeratin network in bile formation and resistance to bile acid challenge and cholestasis in mice #

The intermediate filament cytoskeleton of hepatocytes is composed of keratin (K) 8 and K18 and has important mechanical and nonmechanical functions. However, the potential role of the K8/K18 network for proper membrane targeting of hepatocellular adenosine triphosphate-binding cassette transporters and bile formation is unknown. We therefore designed a comparative study in K8 and K18 knockout mice and respective wild-type controls to test the hypothesis that intermediate filaments of hepatocytes play a role in normal bile formation. In addition, we challenged mice either with a 1% cholic acid-supplemented diet or a diet containing the porphyrinogenic xenobiotic 3,5-diethoxycarbonyl-1,4-dihydrocollidine to determine the effect of K8/K18 loss on bile flow/composition and liver injury under different physiological and toxic stress stimuli. Protein expression levels and membrane localization of various transporters and anion exchangers were compared using western blotting and immunofluorescence microscopy, respectively, and bile flow and composition were determined under various experimental conditions. Our results demonstrate that loss of the intermediate filament network had no significant effect on bile formation and composition, as well as expression levels and membrane targeting of key hepatobiliary transporters under baseline and stress conditions. However, loss of K8 significantly increased liver injury in response to toxic stress. The intermediate filament network of hepatocytes is not specifically required for proper bile formation in mice.

Keratins modulate hepatic cell adhesion, size and G1/S transition

Keratins (Ks) are the intermediate filament (IF) proteins of epithelial cells. Hepatocyte IFs are made solely of keratins 8 and 18 (K8/K18), the hallmark of all simple epithelia. While K8/K18 are essential for maintaining structural integrity, there is accumulating evidence indicating that they also exert non-mechanical functions. We have reported recently that K8/K18-free hepatocytes from K8-null mice are more sensitive to Fas-mediated apoptosis, in line with an increased Fas density at the cell surface and an altered c-Flip regulation of the anti-apoptotic ERK1/2 signaling pathway. In the present study, we show that K8-null hepatocytes attach more rapidly but spread more slowly on a fibronectin substratum and undergo a more efficient G1/S transition than wild-type hepatocytes. Moreover, plectin, an IF associated protein, receptor for activated C kinase 1 (RACK1), a plectin partner, and vinculin, a key component of focal adhesions, distribute differently in spreading K8-null hepatocytes. Cell seeding leads to no differential activation of ERK1/2 in WT versus K8-null hepatocytes, whereas a stronger Akt activation is detected in K8-null hepatocytes. Insulin stimulation also leads to a differential Akt activation, implying altered Akt signaling capacity as a result of the K8/K18 loss. In addition, a delayed autophosphorylation of FAK, a target for integrin β1 signaling, was obtained in seeding K8-null hepatocytes. These alterations in cell cycle-related events in hepatocytes in primary culture are also found in a K8-knockdown H4-II-E-C3 rat hepatoma cell line. Besides, K8/K18-free cells are smaller and exhibit a reduced rate of protein synthesis. In addition, a distinctive cyclin interplay is observed in these K8/K18-free hepatic cells, namely a more efficient cyclin A-dependent G1/S phase transition. Furthermore, K8 re-expression in these cells, following transfer of a human K8 cDNA, restores proper cell size, spreading and growth. Together, these results suggest new interrelated signaling roles of K8/18 with plectin/RACK1 in the modulation of cell attachment/spreading, size/protein synthesis and G1/S transition.

Revival of Bone Strength: The Bottom Line

The continual innovation and proliferation of new research tools for the evaluation and characterization of the skeleton has greatly broadened our understanding of bone physiology and pathology. As the introduction of bone densitometry some 40 years ago was claimed to revolutionize skeletal research—through enabling a quick, precise, and easy noninvasive assessment of bone mineral in vivo—it is quite impossible to predict the potential of the most recent methodological advances in imaging techniques and molecular biology. Today, we are not only able to characterize complex microstructural features of even individual trabeculae, but also to evaluate activities of different bone cells within bone. Unfortunately, it seems at times that the methodological surge has occurred at the cost of studies becoming method-driven, instead of being hypothesis-driven— seeking the true biological mechanisms and relationships. Although whole bone strength testing is among the first methods used to evaluate bone mechanical characteristics, it has not become obsolete. (Whole bone strength test pertains to single mechanical testing of whole bones or excised, complete anatomic bone structures [e.g., proximal femur region] until failure and includes threeor four-point bending tests of long bones and compression tests of proximal femur or vertebrae or columns of vertebrae. The main focus of this analysis is solely on the failure load [strength] of the given bone, whereas structural rigidity [stiffness], postyield behavior, and fatigue characteristics are considered secondary in this respect.) Despite the fact that the nonmechanical functions of the skeleton—hematopoiesis and participation in mineral homeostasis—are chiefly attracting the researchers in the field of osteoporosis, we should never forget that the primary function of the skeleton is locomotion of the body and that only adequately rigid and strong bones make this vital function feasible. It is well established that bones somehow perceive loading-induced strains within the structure and gradually adapt the structure through changes in size, shape, or internal architecture to the prevalent loading environment. Whereas the precise spatial location of each bone element is apparently quite trivial to the above noted subsidiary functions, it can be critical in terms of whole bone strength, “the bottom line.” If we do not know whether the bone as an organ has truly strengthened, we have no certainty of knowing whether the possible changes in any of the intermediate or surrogate measures of bone strength denote only a transient phenomenon–like a “snapshot” of a dynamic movement eventually fading away—or actually a strengthened bone structure as a response to the stimulus of interest. This notion necessitates one to broaden the scope from the mere cellular or tissue-level inspection to the evaluation of bones as structures. The following example from sports shows our concern regarding what we consider the somewhat skewed focus of current experimental skeletal research. We all know that the current technology allows us to measure virtually any imaginable physical characteristic during the course of a long jump (e.g., the length and frequency of the stride of the jumper, athlete’s acceleration or velocity at any given phase during the jump, the height of the jump, or even the oxygen consumption of the muscle activity). However, none of these measures separately or in conjunction tell us the absolute final length of the jump, which is the “bottom line” regarding the athletic performance. Analogous to this, with modern devices, we can readily determine the mineral content, volumetric density, size, shape, and an arsenal of other characteristics of whole bones or even individual bone trabeculae at reasonable precision and accuracy, but at the end, what do we know about the whole bone strength with this information alone? There are infinite ways to construct a bone that has a similar strength but a discernible structure. Needless to say, any change in the rate of bone formation or resorption, irrespective of the underlying cause, ultimately translates into microscopic or macroscopic changes in the bone structure, and possibly, but not necessarily, into the mechanical competence of the whole bone. In the end, it is the whole bone strength that effectively covers virtually all of the individual variability that may be instantaneously, sporadically or permanently present in dynamic/static histomorphometry or in structural particulars (size and shape of the bone, cortical thickness and specific cortical geometry, trabecular architecture, etc.), providing the ultimate assay on bone functional capacity. In 2001, van der Meulen et al. stated persuasively that “the skeletal functional integrity can only be assessed by structural strength tests that measure how well the whole bone can bear load—there is no alternative to testing whole

Evaluation of mechanical materials properties by means of surface micromachined structures

For all micromechanical devices, mechanical properties such as elasticity constants, internal stresses, fracture limits and, for ductile materials, yield limits and strain-hardening behaviour, are of paramount importance in design and use. Furthermore, mechanical integrity aspects are important also for devices with non-mechanical functions. During the last two decades, a diversified and somewhat scrubby flora of methods, instrumentation and microstructures for mechanical microcharacterisation has evolved. Early results were difficult to reproduce and verify, but lately an increasing degree of agreement between reported results is discernible. This paper reviews some recent development of techniques using surface micromachined structures, with special emphasis on stress–strain, internal stress, and fracture property characterisation of brittle and ductile materials. Particular attention is given to evaluation issues such as analytical and numerical modelling (FEA), identification and elimination of error sources, and proper statistical treatment of the results (such as Weibull analysis of fracture results).