The cytoskeleton of the cell is constantly exposed to physical forces that regulate cellular functions. Selected members of the LIM (Lin-11, Isl-1, and Mec-3) domain-containing protein family accumulate along force-bearing actin fibers, with evidence supporting that the LIM domain is solely responsible for this force-induced interaction. However, LIM domain\'s force-induced interactions are not limited to actin. LIMK1 and LMO1, both containing only two tandem LIM domains, are recruited to force-bearing keratin fibers in epithelial cells. This unique recruitment is mediated by their LIM domains and regulated by the sequences outside the LIM domains. Based on in vitro reconstitution of this interaction, LIMK1 and LMO1 directly interact with stretched keratin 8/18 fibers. These results show that LIM domain\'s mechano-sensing abilities extend to the keratin cytoskeleton, highlighting the diverse role of LIM proteins in force-regulated signaling.

Islet β-cell dysfunction is an underlying factor for type I diabetes (T1D) development. Insulin sensing and secretion are tightly regulated in β-cells at multiple subcellular levels. The epithelial intermediate filament (IF) protein keratin (K) 8 is the main β-cell keratin, constituting the filament network with K18. To identify the cell-autonomous functions of K8 in β-cells, mice with targeted deletion of β-cell K8 (K8flox/flox; Ins-Cre) were analyzed for islet morphology, ultrastructure, and integrity, as well as blood glucose regulation and streptozotocin (STZ)-induced diabetes development. Glucose transporter 2 (GLUT2) localization was studied in β-cells in vivo and in MIN6 cells with intact or disrupted K8/K18 filaments. Loss of β-cell K8 leads to a major reduction in K18. Islets without β-cell K8 are more fragile, and these β-cells display disjointed plasma membrane organization with less membranous E-cadherin and smaller mitochondria with diffuse cristae. Lack of β-cell K8 also leads to a reduced glucose-stimulated insulin secretion (GSIS) response in vivo, despite undisturbed systemic blood glucose regulation. K8flox/flox, Ins-Cre mice have a decreased sensitivity to STZ compared with K8 wild-type mice, which is in line with decreased membranous GLUT2 expression observed in vivo, as GLUT2 is required for STZ uptake in β-cells. In vitro, MIN6 cell plasma membrane GLUT2 is rescued in cells overexpressing K8/K18 filaments but mistargeted in cells with disrupted K8/K18 filaments. β-Cell K8 is required for islet and β-cell structural integrity, normal mitochondrial morphology, and GLUT2 plasma membrane targeting, and has implications on STZ sensitivity as well as systemic insulin responses.NEW & NOTEWORTHY Keratin 8 is the main cytoskeletal protein in the cytoplasmic intermediate filament network in β-cells. Here for the first time, we assessed the β-cell autonomous mechanical and nonmechanical roles of keratin 8 in β-cell function. We demonstrated the importance of keratin 8 in islet and β-cell structural integrity, maintaining mitochondrial morphology and GLUT2 plasma membrane targeting.

Here, we describe pathological events potentially involved in the disease pathogenesis of Alexander disease (AxD). This is a primary genetic disorder of astrocyte caused by dominant gain-of-function mutations in the gene coding for an intermediate filament protein glial fibrillary acidic protein (GFAP). Pathologically, this disease is characterized by the upregulation of GFAP and its accumulation as Rosenthal fibers. Although the genetic basis linking GFAP mutations with Alexander disease has been firmly established, the initiating events that promote GFAP accumulation and the role of Rosenthal fibers (RFs) in the disease process remain unknown. Here, we investigate the hypothesis that disease-associated mutations promote GFAP aggregation through aberrant posttranslational modifications. We found high molecular weight GFAP species in the RFs of AxD brains, indicating abnormal GFAP crosslinking as a prominent pathological feature of this disease. In vitro and cell-based studies demonstrate that cystine-generating mutations promote GFAP crosslinking by cysteine-dependent oxidation, resulting in defective GFAP assembly and decreased filament solubility. Moreover, we found GFAP was ubiquitinated in RFs of AxD patients and rodent models, supporting this modification as a critical factor linked to GFAP aggregation. Finally, we found that arginine could increase the solubility of aggregation-prone mutant GFAP by decreasing its ubiquitination and aggregation. Our study suggests a series of pathogenic events leading to AxD, involving interplay between GFAP aggregation and abnormal modifications by GFAP ubiquitination and oxidation. More important, our findings provide a basis for investigating new strategies to treat AxD by targeting abnormal GFAP modifications.

Desmin, the most abundant intermediate filament in cardiomyocytes, plays a key role in maintaining cardiomyocyte structure by interconnecting intracellular organelles, and facilitating cardiomyocyte interactions with the extracellular matrix and neighboring cardiomyocytes. As a consequence, mutations in the desmin gene (DES) can lead to desminopathies, a group of diseases characterized by variable and often severe cardiomyopathies along with skeletal muscle disorders. The basic desmin intermediate filament structure is composed of four segments separated by linkers that further assemble into dimers, tetramers and eventually unit-length filaments that compact radially to give the final form of the filament. Each step in this process is critical for proper filament formation and allow specific interactions within the cell. Mutations within the desmin gene can disrupt filament formation, as seen by aggregate formation, and thus have severe cardiac and skeletal outcomes, depending on the locus of the mutation. The focus of this review is to outline the cardiac molecular consequences of mutations located in the C-terminal part of segment 2B. This region is crucial for ensuring proper desmin filament formation and is a known hotspot for mutations that significantly impact cardiac function.

The dominant structural feature of intermediate filament (IF) proteins is a centrally located α-helix. These long α-helical segments become paired in a parallel orientation to form coiled-coil dimers. Pairs of dimers further coalesce in an anti-parallel orientation to form tetramers. These early stages of intermediate filament assembly can be accomplished solely by the central α-helices. By contrast, the assembly of tetramers into mature intermediate filaments is reliant upon an N-terminal head domain. IF head domains measure roughly 100 amino acids in length and have long been understood to exist in a state of structural disorder. Here, we describe experiments favoring the unexpected idea that head domains self-associate to form transient structural order in the form of labile cross-β interactions. We propose that this weak form of protein structure allows for dynamic regulation of IF assembly and disassembly. We further offer that what we have learned from studies of IF head domains may represent a simple, unifying template for understanding how thousands of other intrinsically disordered proteins help to establish dynamic morphological order within eukaryotic cells.

Associations between actin filaments (AFs) and intermediate filaments (IFs) are frequently observed in living cells. The crosstalk between these cytoskeletal components underpins cellular organization and dynamics; however, the molecular basis of filamentous interactions is not fully understood. Here, we describe the mode of interaction between AFs and desmin IFs (DIFs) in a reconstituted in vitro system.
AFs (rabbit skeletal muscle) and DIFs (chicken gizzard) were labeled with fluorescent dyes. DIFs were immobilized on a heavy meromyosin (HMM)-coated collodion surface. HMM-driven AFs with ATP hydrolysis was assessed in the presence of DIFs. Images of single filaments were obtained using fluorescence microscopy. Vector changes in the trajectories of single AFs were calculated from microscopy images.
AF speed transiently decreased upon contact with DIF. The difference between the incoming and outgoing angles of a moving AF broadened upon contact with a DIF. A smaller incoming angle tended to result in a smaller outgoing angle in a nematic manner. The percentage of moving AFs decreased with an increasing DIF density, but the speed of the moving AFs was similar to that in the no-desmin control. An abundance of DIFs tended to exclude AFs from the HMM-coated surfaces.
DIFs agitate the movement of AFs with the orientation. DIFs can bind to HMMs and weaken actin-myosin interactions.
The study indicates that apart from the binding strength, the accumulation of weak interactions characteristic of filamentous structures may affect the dynamic organization of cell architecture.

ARL15, a small GTPase protein, was linked to metabolic traits in association studies. We aimed to test the Arl15 gene as a functional candidate for metabolic traits in the mouse. CRISPR/Cas9 germline knockout (KO) of Arl15 showed that homozygotes were postnatal lethal and exhibited a complete cleft palate (CP). Also, decreased cell migration was observed from Arl15 KO mouse embryonic fibroblasts (MEFs). Metabolic phenotyping of heterozygotes showed that females had reduced fat mass on a chow diet from 14 weeks of age. Mild body composition phenotypes were also observed in heterozygous mice on a high-fat diet (HFD)/low-fat diet (LFD). Females on a HFD showed reduced body weight, gonadal fat depot weight and brown adipose tissue (BAT) weight. In contrast, in the LFD group, females showed increased bone mineral density (BMD), while males showed a trend toward reduced BMD. Clinical biochemistry analysis of plasma on HFD showed transient lower adiponectin at 20 weeks of age in females. Urinary and plasma Mg2+ concentrations were not significantly different. Our phenotyping data showed that Arl15 is essential for craniofacial development. Adult metabolic phenotyping revealed potential roles in brown adipose tissue and bone development.

The Golgi complex comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region in most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi complex with intermediate filaments (IFs). In this study, we show that the Golgi is in close physical proximity to vimentin IFs in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with vimentin IFs. Loss of vimentin and/or GORAB had a modest effect upon Golgi structure at the steady state. The Golgi underwent more rapid disassembly upon chemical disruption with brefeldin A or nocodazole, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin caused reduced Golgi ribbon integrity when cells were cultured on high-stiffness hydrogels, which was exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi complex and suggest a role for GORAB in this process.

Filensin and phakinin are lens fiber cell-specific proteins that constitute the beaded filaments (BFs) that are critical for maintaining lens transparency. In the Shumiya cataract rat, filensin 94 kDa undergoes N- and C-terminal proteolytic processing to give a transient 50 kDa fragment and a final 38 kDa fragment, just before opacification. To characterize the effects of this processing on filensin function, recombinant proteins representing the two filensin fragments, termed Fil(30-416) and Fil(30-369), respectively, were examined. Fil(30-416) lacks the N-terminal 29 amino acids and the C-terminal 248 amino acids. Fil(30-369) lacks the N-terminal 29 residues and the C-terminal 295 residues. In cell-free assembly characterized by electron microscopy, filensin and Fil(30-416) co-polymerized with phakinin and formed rugged, entangled filaments, whereas Fil(30-369) formed only aggregates. In cultured SW-13 and MCF-7 cells expressing fluorescent fusion proteins, filensin and Fil(30-416) co-polymerized with phakinin and formed cytoplasmic sinuous filaments with different widths, while Fil(30-369) gave aggregates. Therefore, while truncation of the N-terminal 29 amino acids did not affect filament formation, truncation of the C-terminal 295 but not the 248 residues resulted in failure of filament formation. These results indicate that the tail B region (residues 370-416) of rat filensin is essential for filament formation with phakinin. Truncation of the tail B region by proteolytic processing in the cataract rat lens might interfere with BF formation and thereby contribute to opacification.

Intermediate filaments (IFs) are major components of the metazoan cytoskeleton. A long-standing debate concerns the question whether IF network organization only reflects or also determines cell and tissue function. Using Caenorhabditis elegans, we have recently described mutants of the mitogen-activated protein kinase (MAPK) SMA-5 which perturb the organization of the intestinal IF cytoskeleton resulting in luminal widening and cytoplasmic invaginations. Besides these structural phenotypes, systemic dysfunctions were also observed. We now identify the IF polypeptide IFB-2 as a highly efficient suppressor of both the structural and functional deficiencies of mutant sma-5 animals by removing the aberrant IF network. Mechanistically, perturbed IF network morphogenesis is linked to hyperphosphorylation of multiple sites throughout the entire IFB-2 molecule. The rescuing capability is IF isotype-specific and not restricted to sma-5 mutants but extends to mutants that disrupt the function of the cytoskeletal linker IFO-1 and the IF-associated protein BBLN-1. The findings provide strong evidence for adverse consequences of the deranged IF networks with implications for diseases that are characterized by altered IF network organization.