Exploring the Enigma: The Role of the Epithelial Protein Lost in Neoplasm in Normal Physiology and Cancer Pathogenesis.

The cytoskeleton plays a pivotal role in maintaining the epithelial phenotype and is vital to several hallmark processes of cancer. Over the past decades, researchers have identified the epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) as a key regulator of cytoskeletal dynamics, cytoskeletal organization, motility, as well as cell growth and metabolism. Dysregulation of EPLIN is implicated in various aspects of cancer progression, such as tumor growth, invasion, metastasis, and therapeutic resistance. Its altered expression levels or activity can disrupt cytoskeletal dynamics, leading to aberrant cell motility and invasiveness characteristic of malignant cells. Moreover, the involvement of EPLIN in cell growth and metabolism underscores its significance in orchestrating key processes essential for cancer cell survival and proliferation. This review provides a comprehensive exploration of the intricate roles of EPLIN across diverse cellular processes in both normal physiology and cancer pathogenesis. Additionally, this review discusses the possibility of EPLIN as a potential target for anticancer therapy in future studies.

Nucleocytoplasmic transport rates are regulated by cellular processes that modulate GTP availability.

Nucleocytoplasmic transport (NCT), the facilitated diffusion of cargo molecules between the nucleus and cytoplasm through nuclear pore complexes (NPCs), enables numerous fundamental eukaryotic cellular processes. Ran GTPase uses cellular energy in the direct form of GTP to create a gradient across the nuclear envelope (NE) that drives the majority of NCT. We report here that changes in GTP availability resulting from altered cellular physiology modulate the rate of NCT, as monitored using synthetic and natural cargo, and the dynamics of Ran itself. Cell migration, cell spreading, and/or modulation of the cytoskeleton or its connection to the nucleus alter GTP availability and thus rates of NCT, regulating RNA export and protein synthesis. These findings support a model in which changes in cellular physiology that alter GTP availability can regulate the rate of NCT, impacting fundamental cellular processes that extensively utilize NCT.

Semmaphorin 3 A causes immune suppression by inducing cytoskeletal paralysis in tumour-specific CD8+ T cells.

Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8+ T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4+ and CD8+ T cells enhance CD8+ T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8+ T-cell infiltration. We further show that SEMA3A affects CD8+ T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8+ T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours.

Mechanically operated signalling scaffolds.

Cellular signalling is a complex process and involves cascades of enzymes that, in response to a specific signal, give rise to exact cellular responses. Signalling scaffold proteins organise components of these signalling pathways in space and time to co-ordinate signalling outputs. In this review we introduce a new class of mechanically operated signalling scaffolds that are built into the cytoskeletal architecture of the cell. These proteins contain force-dependent binary switch domains that integrate chemical and mechanical signals to introduce quantised positional changes to ligands and persistent alterations in cytoskeletal architecture providing mechanomemory capabilities. We focus on the concept of spatial organisation, and how the cell organises signalling molecules at the plasma membrane in response to specific signals to create order and distinct signalling outputs. The dynamic positioning of molecules using binary switches adds an additional layer of complexity to the idea of scaffolding. The switches can spatiotemporally organise enzymes and substrates dynamically, with the introduction of ∼50 nm quantised steps in distance between them as the switch patterns change. Together these different types of signalling scaffolds and the proteins engaging them, provide a way for an ordering of molecules that extends beyond current views of the cell.

Principles of organelle positioning in motile and non-motile cells.

Cells are equipped with asymmetrically localised and functionally specialised components, including cytoskeletal structures and organelles. Positioning these components to specific intracellular locations in an asymmetric manner is critical for their functionality and affects processes like immune responses, tissue maintenance, muscle functionality, and neurobiology. Here, we provide an overview of strategies to actively move, position, and anchor organelles to specific locations. By conceptualizing the cytoskeletal forces and the organelle-to-cytoskeleton connectivity, we present a framework of active positioning of both membrane-enclosed and membrane-less organelles. Using this framework, we discuss how different principles of force generation and organelle anchorage are utilised by different cells, such as mesenchymal and amoeboid cells, and how the microenvironment influences the plasticity of organelle positioning. Given that motile cells face the challenge of coordinating the positioning of their content with cellular motion, we particularly focus on principles of organelle positioning during migration. In this context, we discuss novel findings on organelle positioning by anchorage-independent mechanisms and their advantages and disadvantages in motile as well as stationary cells.

The α-tubulin acetyltransferase ATAT1: structure, cellular functions, and its emerging role in human diseases.

The acetylation of α-tubulin on lysine 40 is a well-studied post-translational modification which has been associated with the presence of long-lived stable microtubules that are more resistant to mechanical breakdown. The discovery of α-tubulin acetyltransferase 1 (ATAT1), the enzyme responsible for lysine 40 acetylation on α-tubulin in a wide range of species, including protists, nematodes, and mammals, dates to about a decade ago. However, the role of ATAT1 in different cellular activities and molecular pathways has been only recently disclosed. This review comprehensively summarizes the most recent knowledge on ATAT1 structure and substrate binding and analyses the involvement of ATAT1 in a variety of cellular processes such as cell motility, mitosis, cytoskeletal organization, and intracellular trafficking. Finally, the review highlights ATAT1 emerging roles in human diseases and discusses ATAT1 potential enzymatic and non-enzymatic roles and the current efforts in developing ATAT1 inhibitors.

Cooperation of Various Cytoskeletal Components Orchestrates Intercellular Spread of Mitochondria between B-Lymphoma Cells through Tunnelling Nanotubes.

Membrane nanotubes (NTs) are dynamic communication channels connecting spatially separated cells even over long distances and promoting the transport of different cellular cargos. NTs are also involved in the intercellular spread of different pathogens and the deterioration of some neurological disorders. Transport processes via NTs may be controlled by cytoskeletal elements. NTs are frequently observed membrane projections in numerous mammalian cell lines, including various immune cells, but their functional significance in the 'antibody factory' B cells is poorly elucidated. Here, we report that as active channels, NTs of B-lymphoma cells can mediate bidirectional mitochondrial transport, promoted by the cooperation of two different cytoskeletal motor proteins, kinesin along microtubules and myosin VI along actin, and bidirectional transport processes are also supported by the heterogeneous arrangement of the main cytoskeletal filament systems of the NTs. We revealed that despite NTs and axons being different cell extensions, the mitochondrial transport they mediate may exhibit significant similarities. Furthermore, we found that microtubules may improve the stability and lifespan of B-lymphoma-cell NTs, while F-actin strengthens NTs by providing a structural framework for them. Our results may contribute to a better understanding of the regulation of the major cells of humoral immune response to infections.

Dynamic interplay of microtubule and actomyosin forces drive tissue extension.

In order to shape a tissue, individual cell-based mechanical forces have to be integrated into a global force pattern. Over the last decades, the importance of actomyosin contractile arrays, which are the key constituents of various morphogenetic processes, has been established for many tissues. Recent studies have demonstrated that the microtubule cytoskeleton mediates folding and elongation of the epithelial sheet during Drosophila morphogenesis, placing microtubule mechanics on par with actin-based processes. While these studies establish the importance of both cytoskeletal systems during cell and tissue rearrangements, a mechanistic understanding of their functional hierarchy is currently missing. Here, we dissect the individual roles of these two key generators of mechanical forces during epithelium elongation in the developing Drosophila wing. We show that wing extension, which entails columnar-to-cuboidal cell shape remodeling in a cell-autonomous manner, is driven by anisotropic cell expansion caused by the remodeling of the microtubule cytoskeleton from apico-basal to planarly polarized. Importantly, cell and tissue elongation is not associated with Myosin activity. Instead, Myosin II exhibits a homeostatic role, as actomyosin contraction balances polarized microtubule-based forces to determine the final cell shape. Using a reductionist model, we confirm that pairing microtubule and actomyosin-based forces is sufficient to recapitulate cell elongation and the final cell shape. These results support a hierarchical mechanism whereby microtubule-based forces in some epithelial systems prime actomyosin-generated forces.

A Preliminary Investigation of the Roles of Endometrial Cells in Endometriosis Development via In Vitro and In Vivo Analyses.

Endometriosis is a complex gynecological disease that affects more than 10% of women in their reproductive years. While surgery can provide temporary relief from women's pain, symptoms often return in as many as 75% of cases within two years. Previous literature has contributed to theories about the development of endometriosis; however, the exact pathogenesis and etiology remain elusive. We conducted a preliminary investigation into the influence of primary endometrial cells (ECs) on the development and progression of endometriosis. In vitro studies, they were involved in inducing Lipopolysaccharide (LPS) in rat-isolated primary endometrial cells, which resulted in increased nuclear factor-kappa B (NF-κB) and vascular endothelial growth factor (VEGF) mRNA gene expression (quantitative polymerase chain reaction analysis, qPCR) and protein expression (western blot analysis). Additionally, in vivo studies utilized autogenic and allogeneic transplantations (rat to rat) to investigate endometriosis-like lesion cyst size, body weight, protein levels (immunohistochemistry), and mRNA gene expression. These studies demonstrated that estrogen upregulates the gene and protein regulation of cytoskeletal (CK)-18, transforming growth factor-ß (TGF-ß), VEGF, and tumor necrosis factor (TNF)-α, particularly in the peritoneum. These findings may influence cell proliferation, angiogenesis, fibrosis, and inflammation markers. Consequently, this could exacerbate the occurrence and progression of endometriosis.

Contribution of microscopy to a better understanding of the anatomy of pathogenic protists.

In this Inaugural Article the author briefly revises its scientific career and how he starts to work with parasitic protozoa. Emphasis is given to his contribution to topics such as a) the structural organization of the surface of protozoa using freeze-fracture and deep-etching; b) the cytoskeleton of protozoa, especially structures such as the subpellicular microtubules of trypanosomatids, the conoid of Toxoplasma gondii, microtubules and inner membrane complex of this protozoan, and the costa of Tritrichomonas foetus; c) the flagellulm of trypanosomatids, that in addition to the axoneme contains a complex network of filaments that constitute the paraflagellar rod; d) special organelles such as the acidocalcisome, hydrogenosome, and glycosome; and e) the highly polarized endocytic pathway found in epimastigote forms of Trypanosoma cruzi.

Cytoskeletal rearrangement precedes nucleolar remodeling during adipogenesis.

Differentiation of adipose progenitor cells into mature adipocytes entails a dramatic reorganization of the cellular architecture to accommodate lipid storage into cytoplasmic lipid droplets. Lipid droplets occupy most of the adipocyte volume, compressing the nucleus beneath the plasma membrane. How this cellular remodeling affects sub-nuclear structure, including size and number of nucleoli, remains unclear. We describe the morphological remodeling of the nucleus and the nucleolus during in vitro adipogenic differentiation of primary human adipose stem cells. We find that cell cycle arrest elicits a remodeling of nucleolar structure which correlates with a decrease in protein synthesis. Strikingly, triggering cytoskeletal rearrangements mimics the nucleolar remodeling observed during adipogenesis. Our results point to nucleolar remodeling as an active, mechano-regulated mechanism during adipogenic differentiation and demonstrate a key role of the actin cytoskeleton in defining nuclear and nucleolar architecture in differentiating human adipose stem cells.

Kinetic trapping organizes actin filaments within liquid-like protein droplets.

Several actin-binding proteins (ABPs) phase separate to form condensates capable of curating the actin network shapes. Here, we use computational modeling to understand the principles of actin network organization within VASP condensate droplets. Our simulations reveal that the different actin shapes, namely shells, rings, and mixture states are highly dependent on the kinetics of VASP-actin interactions, suggesting that they arise from kinetic trapping. Specifically, we show that reducing the residence time of VASP on actin filaments reduces degree of bundling, thereby promoting assembly of shells rather than rings. We validate the model predictions experimentally using a VASP-mutant with decreased bundling capability. Finally, we investigate the ring opening within deformed droplets and found that the sphere-to-ellipsoid transition is favored under a wide range of filament lengths while the ellipsoid-to-rod transition is only permitted when filaments have a specific range of lengths. Our findings highlight key mechanisms of actin organization within phase-separated ABPs.

Reconstitution of Neuronal Motor Traffic on Septin-Associated Microtubules.

Reconstitution of intracellular transport in cell-free in vitro assays enables the understanding and dissection of the molecular mechanisms that underlie membrane traffic. Using total internal reflection fluorescence (TIRF) microscopy and microtubules, which are immobilized to a functionalized glass surface, the kinetic properties of single kinesin molecules can be imaged and analyzed in the presence or absence of microtubule-associated proteins. Here, we describe methods for the in vitro reconstitution of the motility of the neuronal kinesin motor KIF1A on microtubules associated with heteromeric septin (SEPT2/6/7) complexes. This method can be adapted for various neuronal septin complexes and kinesin motors, leading to new insights into the spatial regulation of neuronal membrane traffic by microtubule-associated septins.

Two new species of the genus Sectonema Thorne, 1930 (Nematoda, Dorylaimida, Aporcelaimidae) from Iran, with new insights into its evolutionary relationships.

Two new species of the genus Sectonema found in northern Iran are characterized, including morphological descriptions and molecular (18S-, 28S-rDNA) analyses. Sectonema tehranense sp. nov. is distinguished by its 7.22 - 8.53 mm long body, lip region offset by constriction and 24 - 31 µm wide with perioral lobes and abundant setae- or cilia-like projections covering the oral field, mural tooth 15.5 - 17 µm long at its ventral side, neck 1091 - 1478 µm long, pharyngeal expansion occupying 61 - 71% of the total neck length, female genital system diovarian, uterus simple and 3.9 - 4.2 times the corresponding body diameter long, transverse vulva (V = 49 - 59), tail short and rounded (44 - 65 µm, c = 99 - 162, c' = 0.6 - 0.8), spicules 111 - 127 µm long, and 7 - 10 spaced ventromedian supplements with hiatus. Sectonema noshahrense sp. nov. displays a 4.07 - 4.73 mm long body, lip region offset by constriction and 23 - 25 µm wide with perioral lobes and abundant setae- or cilia-like projections covering the oral field, odontostyle 14 - 14.5 µm long, neck 722 - 822 µm long, pharyngeal expansion occupying 66 - 68% of the total neck length, female genital system diovarian, uterus simple and 2.4 - 2.7 times the corresponding body diameter long, transverse vulva (V = 54 - 55), tail convex conoid (39 - 47 µm, c = 91 - 111, c' = 0.8 - 0.9), spicules 82 µm long, and seven spaced ventromedian supplements with hiatus. Molecular analyses confirm a maximally supported (Epacrolaimus + Metaporcelaimus + Sectonema) clade and a tentative biogeographical pattern, with sequences of Indolamayan taxa forming a clade separated from those of Palearctic ones. Parallel or convergent evolution processes might be involved in the phylogeny of the species currently classified under Sectonema. This genus is certainly more heterogeneous than previously assumed.

Bacterial Pathogenesis: Assessment of Intracellular Positioning of Pathogen-Containing Vacuoles During Infection.

Intracellular bacterial pathogens implement a diverse array of strategies to target host cells and establish infection. For vacuolar pathogens, the process of pathogen-containing vacuole movement within host cells, termed intracellular trafficking, is central to both pathogen survival and infection progression. Typically a process mediated by secreted virulence factors that manipulate the host cytoskeletal machinery, internalized pathogen-containing vacuoles traffic to the site of replication to establish a unique replicative niche, and if applicable, traffic back toward the host cell periphery for cell-to-cell spread. As such, the intracellular positioning of pathogen-containing vacuoles represents a fundamental measure of infection progression. Here, we describe a fluorescence microscopy-based method to quantitatively assess bacterial intracellular positioning, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This experimental approach can be modified to study infection in diverse host cell types, and with a broad array of pathogens. The system can also be adapted to examine the kinetics of infection, identify secreted virulence factors that mediate host trafficking, investigate host factors that are targeted by the pathogen for trafficking, and assess functional domains within a virulence factor responsible for mediating the phenotype. Collectively, these tools can provide fundamental insight into the pathogenesis of a diverse array of intracellular bacterial pathogens, and new host factors that are hijacked to mediate infection. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture and preparation of host cells Alternate Protocol: Culture and preparation of host cells to assess host factor contribution to bacterial positioning Basic Protocol 2: Infection of epithelial cells with S. Typhimurium Basic Protocol 3: Fluorescence staining for analysis of bacterial positioning Basic Protocol 4: Fluorescence microscopy analysis of bacterial positioning.

The response of Dual-leucine zipper kinase (DLK) to nocodazole: Evidence for a homeostatic cytoskeletal repair mechanism.

Genetic and pharmacological perturbation of the cytoskeleton enhances the regenerative potential of neurons. This response requires Dual-leucine Zipper Kinase (DLK), a neuronal stress sensor that is a central regulator of axon regeneration and degeneration. The damage and repair aspects of this response are reminiscent of other cellular homeostatic systems, suggesting that a cytoskeletal homeostatic response exists. In this study, we propose a framework for understanding DLK mediated neuronal cytoskeletal homeostasis. We demonstrate that low dose nocodazole treatment activates DLK signaling. Activation of DLK signaling results in a DLK-dependent transcriptional signature, which we identify through RNA-seq. This signature includes genes likely to attenuate DLK signaling while simultaneously inducing actin regulating genes. We identify alterations to the cytoskeleton including actin-based morphological changes to the axon. These results are consistent with the model that cytoskeletal disruption in the neuron induces a DLK-dependent homeostatic mechanism, which we term the Cytoskeletal Stress Response (CSR) pathway.

Nesprin proteins: bridging nuclear envelope dynamics to muscular dysfunction.

This review presents a comprehensive exploration of the pivotal role played by the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, with a particular focus on Nesprin proteins, in cellular mechanics and the pathogenesis of muscular diseases. Distinguishing itself from prior works, the analysis delves deeply into the intricate interplay of the LINC complex, emphasizing its indispensable contribution to maintaining cellular structural integrity, especially in mechanically sensitive tissues such as cardiac and striated muscles. Additionally, the significant association between mutations in Nesprin proteins and the onset of Dilated Cardiomyopathy (DCM) and Emery-Dreifuss Muscular Dystrophy (EDMD) is highlighted, underscoring their pivotal role in disease pathogenesis. Through a comprehensive examination of DCM and EDMD cases, the review elucidates the disruptions in the LINC complex, nuclear morphology alterations, and muscular developmental disorders, thus emphasizing the essential function of an intact LINC complex in preserving muscle physiological functions. Moreover, the review provides novel insights into the implications of Nesprin mutations for cellular dynamics in the pathogenesis of muscular diseases, particularly in maintaining cardiac structural and functional integrity. Furthermore, advanced therapeutic strategies, including rectifying Nesprin gene mutations, controlling Nesprin protein expression, enhancing LINC complex functionality, and augmenting cardiac muscle cell function are proposed. By shedding light on the intricate molecular mechanisms underlying nuclear-cytoskeletal interactions, the review lays the groundwork for future research and therapeutic interventions aimed at addressing genetic muscle disorders.

Dynamic localization of the chromosomal passenger complex in trypanosomes is controlled by the orphan kinesins KIN-A and KIN-B.

The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess unique kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles nor how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in Trypanosoma brucei, the kinetoplastid parasite that causes African sleeping sickness. KIN-A and KIN-B form a scaffold for the assembly of the remaining CPC subunits. We show that the C-terminal unstructured tail of KIN-A interacts with the KKT8 complex at kinetochores, while its N-terminal motor domain promotes CPC translocation to spindle microtubules. Thus, the KIN-A:KIN-B complex constitutes a unique 'two-in-one' CPC localization module, which directs the CPC to kinetochores from S phase until metaphase and to the central spindle in anaphase. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora kinases in early eukaryotes.

Pan-microscopic examination of monkeypox virus in trophoblasts cells reveals new insights into virions release through filopodia-like projections.

Vertical transmission has been described following monkeypox virus (MPXV) infection in pregnant women. The presence of MPXV has been reported in the placenta from infected women, but whether pathogens colonize placenta remains unexplored. We identify trophoblasts as a target cell for MPXV replication. In a pan-microscopy approach, we decipher the specific infectious cycle of MPXV and inner cellular structures in trophoblasts. We identified the formation of a specialized region for viral morphogenesis and replication in placental cells. We also reported infection-induced cellular remodeling. We found that MPXV stimulates cytoskeleton reorganization with intercellular extensions for MPXV cell spreading specifically to trophoblastic cells. Altogether, the specific infectious cycle of MPXV in trophoblast cells and these protrusions that were structurally and morphologically similar to filopodia reveal new insights into the infection of MPXV.

Positive and negative durotaxis - mechanisms and emerging concepts.

Cell migration is controlled by the coordinated action of cell adhesion, cytoskeletal dynamics, contractility and cell extrinsic cues. Integrins are the main adhesion receptors to ligands of the extracellular matrix (ECM), linking the actin cytoskeleton to the ECM and enabling cells to sense matrix rigidity and mount a directional cell migration response to stiffness gradients. Most models studied show preferred migration of single cells or cell clusters towards increasing rigidity. This is referred to as durotaxis, and since its initial discovery in 2000, technical advances and elegant computational models have provided molecular level details of stiffness sensing in cell migration. However, modeling has long predicted that, depending on cell intrinsic factors, such as the balance of cell adhesion molecules (clutches) and the motor proteins pulling on them, cells might also prefer adhesion to intermediate rigidity. Recently, experimental evidence has supported this notion and demonstrated the ability of cells to migrate towards lower rigidity, in a process called negative durotaxis. In this Review, we discuss the significant conceptual advances that have been made in our appreciation of cell plasticity and context dependency in stiffness-guided directional cell migration.