Cytoskeletal Architecture Research Articles

Abstract 3348: Unraveling new strategies for early detection of invasive cancer cells: A special focus on the nuclear and cytoskeletal architecture

Abstract Introduction: Cancer invasion is a hallmark of cancer progression and the leading cause of cancer mortality worldwide. However, the identification of invasive cancer cells at an early stage remains challenging. Therefore, there is an urgent need to develop novel biomarkers and innovative strategies to successfully tackle cancer invasion. It is well established that the structure and organization of the nucleus and the cytoskeleton are dynamically orchestrated during many cellular processes, including cancer invasion. Thus, in this study, we investigated fine-tuned nuclear and cytoskeletal patterns associated with invasion, taking into consideration the cell-extracellular matrix (ECM) interaction. Methods: In this study, we took advantage of an ECM Array platform (MicroMatrix 36, MicroStem) and a panel of invasive and non-invasive gastric cancer cells. For validation purposes, we assessed cells transfected with wild type or mutant E-cadherin, which lead to distinct adhesion and invasion phenotypes. A comprehensive analysis of nuclei and cytoskeletal architectural features was performed. Nuclei area, intensity, perimeter, eccentricity, circularity, solidity and entropy were evaluated. Notably, a new computational pipeline was developed to characterize cytoskeletal structures, namely orientation, compactness, radiality, bundling, parallelness, morphology, among others. Results and Discussion: Our results have shown that the invasive abilities of cancer cells are associated with unique nuclear and cytoskeletal modifications. Moreover, we verified that invasive cells adhere preferentially to specific ECM components. Interestingly, we could observe an association between nuclear phenotypes and increased attachment abilities, suggesting that the ECM may modulate nuclear morphometrics thus affecting the invasive behavior of cancer cells. Of note, cell lines harboring E-cadherin mutations corroborated the existence of a nuclear signature associated with invasive potential. Remarkably, the proposed computational framework has shown that the microtubules of cells with disrupted E-cadherin are shorter, have uniform length patterns and are more compactly distributed as compared with cells with wild-type E-cadherin. Conclusion: Overall, this study demonstrates that the use of nuclear and cytoskeletal architectural features could provide an efficient strategy to identify cells with invasive potential. Further investigation and integration of this approach with other cellular properties could have an impact in diagnosis, prognosis and therapeutic strategies. Citation Format: Maria Sofia Fernandes, Joana Figueiredo, Diogo Vieira, Soraia Melo, Ana Margarida Moreira, João Miguel Sanches, Raquel Seruca. Unraveling new strategies for early detection of invasive cancer cells: A special focus on the nuclear and cytoskeletal architecture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3348.

The Zinc-BED Transcription Factor Bedwarfed Promotes Proportional Dendritic Growth and Branching through Transcriptional and Translational Regulation in Drosophila.

Dendrites are the primary points of sensory or synaptic input to a neuron and play an essential role in synaptic integration and neural function. Despite the functional importance of dendrites, relatively less is known about the underlying mechanisms regulating cell type-specific dendritic patterning. Herein, we have dissected the functional roles of a previously uncharacterized gene, CG3995, in cell type-specific dendritic development in Drosophila melanogaster. CG3995, which we have named bedwarfed (bdwf), encodes a zinc-finger BED-type protein that is required for proportional growth and branching of dendritic arbors. It also exhibits nucleocytoplasmic expression and functions in both transcriptional and translational cellular pathways. At the transcriptional level, we demonstrate a reciprocal regulatory relationship between Bdwf and the homeodomain transcription factor (TF) Cut. We show that Cut positively regulates Bdwf expression and that Bdwf acts as a downstream effector of Cut-mediated dendritic development, whereas overexpression of Bdwf negatively regulates Cut expression in multidendritic sensory neurons. Proteomic analyses revealed that Bdwf interacts with ribosomal proteins and disruption of these proteins resulted in phenotypically similar dendritic hypotrophy defects as observed in bdwf mutant neurons. We further demonstrate that Bdwf and its ribosomal protein interactors are required for normal microtubule and F-actin cytoskeletal architecture. Finally, our findings reveal that Bdwf is required to promote protein translation and ribosome trafficking along the dendritic arbor. These findings shed light on the complex, combinatorial, and multi-functional roles of transcription factors (TFs) in directing the diversification of cell type-specific dendritic development.

Isolation and identification of extracellular matrix proteins from oil-based CASPERized mouse brains for matrisomal analysis

The extracellular matrix (ECM) components present within all tissues and organs help to maintain the cytoskeletal architecture and tissue morphology. Although the ECM plays a role in cellular events and signaling pathways, it has not been well studied due its insolubility and complexity. Brain tissue has a higher cell density and weaker mechanical strength than other tissues in the body. When removing cells using a general decellularization method to produce scaffolds and obtain ECM proteins, various problems must be considered because tissues are easily damaged. To retain the brain shape and ECM components, we performed decellularization in combination with polymerization. We immersed mouse brains in oil for polymerization and decellularization via O-CASPER (Oil-based Clinically and Experimentally Applicable Acellular Tissue Scaffold Production for Tissue Engineering and Regenerative Medicine) and then isolated ECM components using sequential matrisome preparation reagents (SMPRs), namely, RIPA, PNGase F, and concanavalin A. Adult mouse brains were preserved with our decellularization method. Western blot and LC-MS/MS analyses revealed that ECM components, including collagen and laminin, were isolated efficiently from decellularized mouse brains using SMPRs. Our method will be useful to obtain matrisomal data and perform functional studies using adult mouse brains and other tissues.

The energetics of rapid cellular mechanotransduction

Cells throughout the human body detect mechanical forces. While it is known that the rapid (millisecond) detection of mechanical forces is mediated by force-gated ion channels, a detailed quantitative understanding of cells as sensors of mechanical energy is still lacking. Here, we combine atomic force microscopy with patch-clamp electrophysiology to determine the physical limits of cells expressing the force-gated ion channels (FGICs) Piezo1, Piezo2, TREK1, and TRAAK. We find that, depending on the ion channel expressed, cells can function either as proportional or nonlinear transducers of mechanical energy and detect mechanical energies as little as ~100 fJ, with a resolution of up to ~1 fJ. These specific energetic values depend on cell size, channel density, and cytoskeletal architecture. We also make the surprising discovery that cells can transduce forces either nearly instantaneously (<1 ms) or with a substantial time delay (~10 ms). Using a chimeric experimental approach and simulations, we show how such delays can emerge from channel-intrinsic properties and the slow diffusion of tension in the membrane. Overall, our experiments reveal the capabilities and limits of cellular mechanosensing and provide insights into molecular mechanisms that different cell types may employ to specialize for their distinct physiological roles.

The energetics of rapid cellular mechanotransduction.

Cells throughout the human body detect mechanical forces. While it is known that the rapid (millisecond) detection of mechanical forces is mediated by force-gated ion channels, a detailed quantitative understanding of cells as sensors of mechanical energy is still lacking. Here, we combine atomic force microscopy with patch-clamp electrophysiology to determine the physical limits of cells expressing the force-gated ion channels Piezo1, Piezo2, TREK1, and TRAAK. We find that, depending on the ion channel expressed, cells can function either as proportional or nonlinear transducers of mechanical energy, detect mechanical energies as little as ∼100 fJ, and with a resolution of up to ∼1 fJ. These specific energetic values depend on cell size, channel density, and cytoskeletal architecture. We also make the surprising discovery that cells can transduce forces either nearly instantaneously (<1ms), or with substantial time delay (∼10 ms). Using a chimeric experimental approach and simulations we show how such delays can emerge from channel-intrinsic properties and the slow diffusion of tension in the membrane. Overall, our experiments reveal the capabilities and limits of cellular mechanosensing and provide insights into molecular mechanisms that different cell types may employ to specialize for their distinct physiological roles.

Cytoskeletal orchestration of glucose uptake in Sertoli cell to support efferocytosis of apoptotic germ cells

Efferocytosis of non-viable germ cells by Sertoli cells (SCs) constitutes a sentinel for testis homeostasis, yet how SCs signal for the metabolic and cytoskeletal adaption to this energetically costly process remains unexplored. Spectrin is membrane-associated periodic skeleton assembled into an actin-spectrin-based cytoskeletal structure with an interaction with glucose transporter Glut1. The contribution of spectrin to glucose uptake and efferocytosis is unknown. In this study, we identified a cross-regulation between glucose metabolism and efferocytosis in SCs. Pharmacological or genetic inhibition of glucose uptake or glycolysis compromises efferocytosis activity. We further found that βII-spectrin is a hitherto unappreciated regulator of glucose metabolism and cytoskeletal architecture. βII-spectrin deficiency impairs glucose uptake and lactate production in SCs. Moreover, a defective assembly of cytoskeleton and a loss of blood-testis barrier integrity are also featured by SCs deficient in βII-spectrin. The disruption in glucose metabolism and cytoskeletal organization synergistically lead to a defective efferocytosis. In vivo siRNA-mediated targeting of βII-spectrin in testis causes an obvious morphological aberration in seminiferous epithelium with the presence of exfoliated germ cells and multinucleated giant cells. Importantly, a decrease in expression of αII/βII-spectrin was observed in testes of Adjudin-induced infertility model. By exploring the functional relevance of βII-spectrin to the metabolic and cytoskeletal regulation of efferocytosis, our study proposes a potential link between βII-spectrin deregulation and male infertility.

A Homozygous PPP1R21 Splice Variant Associated with Severe Developmental Delay, Absence of Speech, and Muscle Weakness Leads to Activated Proteasome Function

PPP1R21 acts as a co-factor for protein phosphatase 1 (PP1), an important serine/threonine phosphatase known to be essential for cell division, control of glycogen metabolism, protein synthesis, and muscle contractility. Bi-allelic pathogenic variants in PPP1R21 were linked to a neurodevelopmental disorder with hypotonia, facial dysmorphism, and brain abnormalities (NEDHFBA) with pediatric onset. Functional studies unraveled impaired vesicular transport as being part of PPP1R21-related pathomechanism. To decipher further the pathophysiological processes leading to the clinical manifestation of NEDHFBA, we investigated the proteomic signature of fibroblasts derived from the first NEDHFBA patient harboring a splice-site mutation in PPP1R21 and presenting with a milder phenotype. Proteomic findings and further functional studies demonstrate a profound activation of the ubiquitin–proteasome system with presence of protein aggregates and impact on cellular fitness and moreover suggest a cross-link between activation of the proteolytic system and cytoskeletal architecture (including filopodia) as exemplified on paradigmatic proteins including actin, thus extending the pathophysiological spectrum of the disease. In addition, the proteomic signature of PPP1R21-mutant fibroblasts displayed a dysregulation of a variety of proteins of neurological relevance. This includes increase proteins which might act toward antagonization of cellular stress burden in terms of pro-survival, a molecular finding which might accord with the presentation of a milder phenotype of our NEDHFBA patient.

A Multi-Trait Association Analysis of Brain Disorders and Platelet Traits Identifies Novel Susceptibility Loci for Major Depression, Alzheimer’s and Parkinson’s Disease

Among candidate neurodegenerative/neuropsychiatric risk-predictive biomarkers, platelet count, mean platelet volume and platelet distribution width have been associated with the risk of major depressive disorder (MDD), Alzheimer's disease (AD) and Parkinson's disease (PD) through epidemiological and genomic studies, suggesting partial co-heritability. We exploited these relationships for a multi-trait association analysis, using publicly available summary statistics of genome-wide association studies (GWASs) of all traits reported above. Gene-based enrichment tests were carried out, as well as a network analysis of significantly enriched genes. We analyzed 4,540,326 single nucleotide polymorphisms shared among the analyzed GWASs, observing 149 genome-wide significant multi-trait LD-independent associations (p < 5 × 10-8) for AD, 70 for PD and 139 for MDD. Among these, 27 novel associations were detected for AD, 34 for PD and 40 for MDD. Out of 18,781 genes with annotated variants within ±10 kb, 62 genes were enriched for associations with AD, 70 with PD and 125 with MDD (p < 2.7 × 10-6). Of these, seven genes were novel susceptibility loci for AD (EPPK1, TTLL1, PACSIN2, TPM4, PIF1, ZNF689, AZGP1P1), two for PD (SLC26A1, EFNA3) and two for MDD (HSPH1, TRMT61A). The resulting network showed a significant excess of interactions (enrichment p = 1.0 × 10-16). The novel genes that were identified are involved in the organization of cytoskeletal architecture (EPPK1, TTLL1, PACSIN2, TPM4), telomere shortening (PIF1), the regulation of cellular aging (ZNF689, AZGP1P1) and neurodevelopment (EFNA3), thus, providing novel insights into the shared underlying biology of brain disorders and platelet parameters.

Delayed reorganisation of F-actin cytoskeleton and reversible chromatin condensation in scleral fibroblasts under simulated pathological strain

Mechanical loading regulates the functional capabilities of the ocular system, particularly in the sclera (‘white of the eye’) – the principal load-bearing tissue of the ocular globe. Resident fibroblasts of the scleral eye wall are continuously subjected to fluctuating mechanical strains arising from eye movements, cerebrospinal fluid pressure and, most influentially, intra-ocular pressure (IOP). Whilst fibroblasts are hypothesised to actively participate in scleral biomechanics, to date limited information has been reported on how the macroscopic stresses and strains are transmitted via their cytoskeletal networks. In this study, the effect of applying either a ‘physiological load’ (simulating healthy IOP) or a ‘pathological load’ (simulating an elevated glaucomatous IOP) to bovine scleral fibroblasts, as a model of human glaucoma, was conducted to characterise cytoskeletal organisation, chromatin condensation and cell dimensions using immunofluorescence confocal microscopy. Quantification of cell parameters and cytoskeletal element anisotropy were subsequently performed using FibrilTool, and chromatin condensation parameter assessment through a bespoke MATLAB script. The novel findings suggest that physiological load-induced F-actin rearrangement is transient, whereas pathological load, recapitulating in vivo glaucomatous IOP levels, had a reversible and inhibitory influence on remodelling of the cytoskeletal architecture and, further, induction of chromatin condensation. Ultimately, this could compromise cell behaviour. These findings could provide valuable insight into the mechanism(s) used by scleral fibroblasts to mechanically adapt to support biomechanical tissue integrity, and how it could be potentially modified for therapeutic avenues targeting mechanically mediated ocular pathologies such as glaucoma.

α-Actinin-4 recruits Shp2 into focal adhesions to potentiate ROCK2 activation in podocytes

Cell-matrix adhesions are mainly provided by integrin-mediated focal adhesions (FAs). We previously found that Shp2 is essential for FA maturation by promoting ROCK2 activation at FAs. In this study, we further delineated the role of α-actinin-4 in the FA recruitment and activation of Shp2. We used the conditional immortalized mouse podocytes to examine the role of α-actinin-4 in the regulation of Shp2 and ROCK2 signaling. After the induction of podocyte differentiation, Shp2 and ROCK2 were strongly activated, concomitant with the formation of matured FAs, stress fibers, and interdigitating intracellular junctions in a ROCK-dependent manner. Gene knockout of α-actinin-4 abolished the Shp2 activation and subsequently reduced matured FAs in podocytes. We also demonstrated that gene knockout of ROCK2 impaired the generation of contractility and interdigitating intercellular junctions. Our results reveal the role of α-actinin-4 in the recruitment of Shp2 at FAs to potentiate ROCK2 activation for the maintenance of cellular contractility and cytoskeletal architecture in the cultured podocytes.

Physiologic and pathophysiologic roles of AKAP12.

A kinase anchoring protein (AKAP) 12 is a scaffolding protein that improves the specificity and efficiency of spatiotemporal signal through assembling intracellular signal proteins into a specific complex. AKAP12 is a negative mitogenic regulator that plays an important role in controlling cytoskeletal architecture, maintaining endothelial integrity, regulating glial function and forming blood-brain barrier (BBB) and blood retinal barrier (BRB). Moreover, elevated or reduced AKAP12 contributes to a variety of diseases. Complex connections between AKAP12 and various diseases including chronic liver diseases (CLDs), inflammatory diseases and a series of cancers will be tried to delineate in this paper. We first describe the expression, distribution and physiological function of AKAP12. Then we summarize the current knowledge of different connections between AKAP12 expression and various diseases. Some research groups have found paradoxical roles of AKAP12 in different diseases and further confirmation is needed. This paper aims to assess the role of AKAP12 in physiology and diseases to help lay the foundation for the design of small molecules for specific AKAP12 to correct the pathological signal defects.

Surface Functionalized Polyaniline Nanofibers:Chitosan Nanocomposite for Promoting Neuronal-like Differentiation of Primary Adipose Derived Mesenchymal Stem Cells and Urease Activity.

Bioscaffolds having electrically conducting polymers (CPs) have become increasingly relevant in tissue engineering (TE) because of their ability to regulate conductivity and promote biological function. With this in mind, the current study shows a conducting polyaniline nanofibers (PNFs) dispersed chitosan (Ch) nanocomposites scaffold with a simple one-step surface functionalization approach using glutaraldehyde for potential neural regeneration applications. According to the findings, 4 wt % PNFs dispersion in Ch matrix is an optimal concentration for achieving desirable biological functions while maintaining required physicochemical properties as evidenced by SEM, XRD, current-voltage (I-V) measurement, mechanical strength test, and in vitro biodegradability test. Surface chemical compositional analysis using XPS and ATR FT-IR confirms the incorporation of aldehyde functionality after functionalization, which is corroborated by surface energy calculations following the Van Oss-Chaudhury-Good method. Surface functionalization induced enhancement in surface hydrophilicity in terms of the polar component of surface energy (γiAB) from 6.35 to 12.54 mN m-1 along with an increase in surface polarity from 13.61 to 22.54%. Functionalized PNF:Ch scaffolds demonstrated improvement in enzyme activity from 67 to 94% and better enzyme kinetics with a reduction of Michaelis constants (Km) from 21.55 to 13.81 mM, indicating favorable protein-biomaterial interactions and establishing them as biologically perceptible materials. Surface functionalization mediated improved cell-biomaterial interactions led to improved viability, adhesion, and spreading of primary adipose derived mesenchymal stem cells (ADMSCs) as well as improved immunocompatibility. Cytoskeletal architecture assessment under differentiating media containing 10 ng/mL of each basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) revealed significant actin remodeling with neurite-like projections on the functionalized scaffolds after 14 days. Immunocytochemistry results showed that more than 85% of cells expressed early neuron specific β III tubulin protein on the functionalized scaffolds, whereas glial fibrillary acidic protein (GFAP) expression was limited to approximately 40% of cells. The findings point to the functionalized nanocomposites' potential as a smart scaffold for electrically stimulated neural regeneration, as they are flexible enough to be designed into microchanneled or conduit-like structures that mimic the microstructures and mechanical properties of peripheral nerves.

How Microtubules Build the Spindle Branch by Branch.

The microtubule (MT) cytoskeleton provides the architecture that governs intracellular organization and the regulated motion of macromolecules through the crowded cytoplasm. The key to establishing a functioning cytoskeletal architecture is regulating when and where new MTs are nucleated. Within the spindle, the vast majority of MTs are generated through a pathway known as branching MT nucleation, which exponentially amplifies MT number in a polar manner. Whereas other MT nucleation pathways generally require a complex organelle such as the centrosome or Golgi apparatus to localize nucleation factors, the branching site is based solely on a simple, preformed MT, making it an ideal system to study MT nucleation. In this review, we address recent developments in characterizing branching factors, the branching reaction, and its regulation, as well as branching MT nucleation in systems beyond the spindle and within human disease.

Unveiling the nanotoxicological aspects of Se nanomaterials differing in size and morphology.

Although the general concept of nanotechnology relies on exploitation of size-dependent properties of nanoscaled materials, the relation between the size/morphology of nanoparticles with their biological activity remains not well understood. Therefore, we aimed at investigating the biological activity of Se nanoparticles, one of the most promising candidates of nanomaterials for biomedicine, possessing the same crystal structure, but differing in morphology (nanorods vs. spherical particles) and aspect ratios (AR, 11.5 vs. 22.3 vs. 1.0) in human cells and BALB/c mice. Herein, we report that in case of nanorod-shaped Se nanomaterials, AR is a critical factor describing their cytotoxicity and biocompatibility. However, spherical nanoparticles (AR 1.0) do not fit this statement and exhibit markedly higher cytotoxicity than lower-AR Se nanorods. Beside of cytotoxicity, we also show that morphology and size substantially affect the uptake and intracellular fate of Se nanomaterials. In line with in vitro data, in vivo i.v. administration of Se nanomaterials revealed the highest toxicity for higher-AR nanorods followed by spherical nanoparticles and lower-AR nanorods. Moreover, we revealed that Se nanomaterials are able to alter intracellular redox homeostasis, and affect the acidic intracellular vesicles and cytoskeletal architecture in a size- and morphology-dependent manner. Although the tested nanoparticles were produced from the similar sources, their behavior differs markedly, since each type is promising for several various application scenarios, and the presented testing protocol could serve as a concept standardizing the biological relevance of the size and morphology of the various types of nanomaterials and nanoparticles.

Understanding the Behaviour of Human Cell Types under Simulated Microgravity Conditions: The Case of Erythrocytes.

Erythrocytes are highly specialized cells in human body, and their main function is to ensure the gas exchanges, O2 and CO2, within the body. The exposure to microgravity environment leads to several health risks such as those affecting red blood cells. In this work, we investigated the changes that occur in the structure and function of red blood cells under simulated microgravity, compared to terrestrial conditions, at different time points using biochemical and biophysical techniques. Erythrocytes exposed to simulated microgravity showed morphological changes, a constant increase in reactive oxygen species (ROS), a significant reduction in total antioxidant capacity (TAC), a remarkable and constant decrease in total glutathione (GSH) concentration, and an augmentation in malondialdehyde (MDA) at increasing times. Moreover, experiments were performed to evaluate the lipid profile of erythrocyte membranes which showed an upregulation in the following membrane phosphocholines (PC): PC16:0_16:0, PC 33:5, PC18:2_18:2, PC 15:1_20:4 and SM d42:1. Thus, remarkable changes in erythrocyte cytoskeletal architecture and membrane stiffness due to oxidative damage have been found under microgravity conditions, in addition to factors that contribute to the plasticity of the red blood cells (RBCs) including shape, size, cell viscosity and membrane rigidity. This study represents our first investigation into the effects of microgravity on erythrocytes and will be followed by other experiments towards understanding the behaviour of different human cell types in microgravity.

Emerging interplay of cytoskeletal architecture, cytomechanics and pluripotency.

Pluripotent stem cells (PSCs) are capable of differentiating into all three germ layers and trophoblasts, whereas tissue-specific adult stem cells have a more limited lineage potency. Although the importance of the cytoskeletal architecture and cytomechanical properties in adult stem cell differentiation have been widely appreciated, how they contribute to mechanotransduction in PSCs is less well understood. Here, we discuss recent insights into the interplay of cellular architecture, cell mechanics and the pluripotent states of PSCs. Notably, the distinctive cytomechanical and morphodynamic profiles of PSCs are accompanied by a number of unique molecular mechanisms. The extent to which such mechanobiological signatures are intertwined with pluripotency regulation remains an open question that may have important implications in developmental morphogenesis and regenerative medicine.

APOLD1 loss causes endothelial dysfunction involving cell junctions, cytoskeletal architecture, and Weibel-Palade bodies, while disrupting hemostasis.

Vascular homeostasis is impaired in various diseases thereby contributing to the progression of their underlying pathologies. The endothelial immediate early gene Apolipoprotein L domain-containing 1 (APOLD1) helps to regulate endothelial function. However, its precise role in endothelial cell biology remains unclear. We have localized APOLD1 to endothelial cell contacts and to Weibel-Palade bodies (WPB) where it associates with von Willebrand factor (VWF) tubules. Silencing of APOLD1 in primary human endothelial cells disrupted the cell junction-cytoskeletal interface, thereby altering endothelial permeability accompanied by spontaneous release of WPB contents. This resulted in an increased presence of WPB cargoes, notably VWF and angiopoietin-2 in the extracellular medium. Autophagy flux, previously recognized as an essential mechanism for the regulated release of WPB, was impaired in the absence of APOLD1. In addition, we report APOLD1 as a candidate gene for a novel inherited bleeding disorder across three generations of a large family in which an atypical bleeding diathesis was associated with episodic impaired microcirculation. A dominant heterozygous nonsense APOLD1:p.R49* variant segregated to affected family members. Compromised vascular integrity resulting from an excess of plasma angiopoietin-2, and locally impaired availability of VWF may explain the unusual clinical profile of APOLD1:p.R49* patients. In summary, our findings identify APOLD1 as an important regulator of vascular homeostasis and raise the need to consider testing of endothelial cell function in patients with inherited bleeding disorders without apparent platelet or coagulation defects.

TREPs-A New Class of Functional tRNA-Encoded Peptides.

We asked if transfer RNA (tRNA) ever got an opportunity of translating its own sequence during evolution, what would have been the function of such tRNA-encoded peptides (tREPs)? If not, could one artificially synthesize tREPs to study the corresponding functional outcomes? Here, we report a novel, first-in-the-class, chemically synthesized tREP-18 molecule originating from the Escherichia coli tRNA sequence showing potent antileishmanial property. As a first step, E. coli tRNAs were computationally translated into peptide sequence equivalents and a database of full-length hypothetical tREPs was created. The tREP sequences were sent into sequence, structure, and energy filters to narrow down potential peptides for experimental validation. Based on the functional predictions, tREPs were screened against antiparasitic targets, leading to the identification of tREP-18 as a potential antiparasitic peptide. The in vitro assay of chemically synthesized tREP-18 on the Ag83 strain of Leishmania donovani showed its potent antileishmanial property (IC50 value of 22.13 nM). The atomic force microscopy and scanning electron microscopy images indicated significant alteration in the cytoskeletal architecture of tREP-18-treated parasites. Also, tREP-18 seems to destabilize the mitochondrial membrane potential of parasites, disrupting their cellular integrity and leading to parasitic death. The cellular assays of the tREP-18 peptide on the BS12 strain, a clinical isolate of post-kala azar dermal leishmaniasis, demonstrated its significant efficacy at an IC50 value of 15 nM. The tREP-18 peptide showed a toxic effect on the amastigote stage of the parasite, showing macrophage pathogen clearance at a concentration of 22.5 nM. This study provides the proof of the concept of making a new class of functional peptides from tRNA sequences. It also opens a huge untapped tRNA-peptide space toward novel discoveries and applications. In the future, it would be interesting to perform tREP edits and redesign tREPs toward specific applications.

SHROOM4 Variants Are Associated With X-Linked Epilepsy With Features of Generalized Seizures or Generalized Discharges.

ObjectiveSHROOM4 gene encodes an actin-binding proteins, which plays an important role in cytoskeletal architecture, synaptogenesis, and maintaining gamma-aminobutyric acid receptors-mediated inhibition. SHROOM4 mutations were reported in patients with the Stocco dos Santos type of X-linked syndromic intellectual developmental disorder (SDSX; OMIM# 300434). In this study, we investigated the association between SHROOM4 and epilepsy.MethodsTrios-based whole-exome sequencing was performed in a cohort of 320 cases with idiopathic generalized epilepsy or idiopathic partial epilepsy. Protein modeling was used to assess the damaging effects of variations.ResultsSix hemizygous missense SHROOM4 variants, including c.13C > A/p. Pro5Thr, c.3236C > T/p.Glu1079Ala, c.3581C > T/p.Ser1194Leu, c.4288C > T/p.Arg1430Cys, c.4303G > A/p.Val1435Met, c.4331C > T/p.Pro1444Leu, were identified in six cases with idiopathic epilepsy without intellectual disability. All patients presented with features of generalized seizures or generalized discharges. These hemizygous variants had no or extremely low allele frequencies in controls and showed statistically higher frequency in the case cohort than controls. All variants were predicted to alter hydrogen bond with surrounding amino acids or decreased protein stability. The SHROOM4 variants reported in patients with SDSX were mostly destructive or duplicative variants; in contrast, the SHROOM4 variants were all missense variants, suggesting a potential genotype-phenotype correlation. The two missense variants associated with SDSX were located in the middle of SHROOM4 protein, whereas variants associated with idiopathic epilepsy were located around the N-terminal PDZ domain and the C-terminal ASD2 domain.SignificanceSHROOM4 was potentially a candidate pathogenic gene of idiopathic epilepsy without intellectual disability. The genotype-phenotype correlation and sub-regional effect helps understanding the mechanism underlying phenotypic variation.

PINCH1 Promotes Fibroblast Migration in Extracellular Matrices and Influences Their Mechanophenotype.

Cell migration performs a critical function in numerous physiological processes, including tissue homeostasis or wound healing after tissue injury, as well as pathological processes that include malignant progression of cancer. The efficiency of cell migration and invasion appears to be based on the mechano-phenotype of the cytoskeleton. The properties of the cytoskeleton depend on internal cytoskeletal and external environmental factors. A reason for this are connections between the cell and its local matrix microenvironment, which are established by cell-matrix adhesion receptors. Upon activation, focal adhesion proteins such as PINCH1 are recruited to sites where focal adhesions form. PINCH1 specifically couples through interactions with ILK, which binds to cell matrix receptors and the actomyosin cytoskeleton. However, the role of PINCH1 in cell mechanics regulating cellular motility in 3D collagen matrices is still unclear. PINCH1 is thought to facilitate 3D motility by regulating cellular mechanical properties, such as stiffness. In this study, PINCH1 wild-type and knock-out cells were examined for their ability to migrate in dense extracellular 3D matrices. Indeed, PINCH1 wild-type cells migrated more numerously and deeper in 3D matrices, compared to knock-out cells. Moreover, cellular deformability was determined, e.g., elastic modulus (stiffness). PINCH1 knock-out cells are more deformable (compliable) than PINCH1 wild-type cells. Migration of both PINCH1−/− cells and PINCH1fl/fl cells was decreased by Latrunculin A inhibition of actin polymerization, suggesting that actin cytoskeletal differences are not responsible for the discrepancy in invasiveness of the two cell types. However, the mechanical phenotype of PINCH1−/− cells may be reflected by Latrunculin A treatment of PINCH1fl/fl cells, as they exhibit resembling deformability to untreated PINCH1−/− cells. Moreover, an apparent mismatch exists between the elongation of the long axis and the contraction of the short axis between PINCH1fl/fl cells and PINCH1−/− cells following Latrunculin A treatment. There is evidence of this indicating a shift in the proxy values for Poisson’s ratio in PINCH1−/− cells compared with PINCH1fl/fl cells. This is probably attributable to modifications in cytoskeletal architecture. The non-muscle myosin II inhibitor Blebbistatin also reduced the cell invasiveness in 3D extracellular matrices but instead caused a stiffening of the cells. Finally, PINCH1 is apparently essential for providing cellular mechanical stiffness through the actin cytoskeleton, which regulates 3D motility.