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20 pages, 6897 KiB

Open AccessArticle

Mechanical Cues, E-Cadherin Expression and Cell “Sociality” Are Crucial Crossroads in Determining Pancreatic Ductal Adenocarcinoma Cells Behavior

by Francesca Bianchi, Michele Sommariva, Laura Brigida Cornaghi, Luca Denti, Ambra Nava, Francesca Arnaboldi, Claudia Moscheni and Nicoletta Gagliano

Cells 2022, 11(8), 1318; https://doi.org/10.3390/cells11081318 - 13 Apr 2022

Cited by 4 | Viewed by 2675

Abstract

E-cadherin, an epithelial-to-mesenchymal transition (EMT) marker, is coupled to actin cytoskeleton and distributes cell forces acting on cells. Since YAP transduces mechanical signals involving actin cytoskeleton, we aimed to investigate the relationship between YAP and mechanical cues in pancreatic ductal adenocarcinoma (PDAC) cell [...] Read more.

E-cadherin, an epithelial-to-mesenchymal transition (EMT) marker, is coupled to actin cytoskeleton and distributes cell forces acting on cells. Since YAP transduces mechanical signals involving actin cytoskeleton, we aimed to investigate the relationship between YAP and mechanical cues in pancreatic ductal adenocarcinoma (PDAC) cell lines, characterized by different EMT-related phenotypes, cultured in 2D monolayers and 3D spheroids. We observed that the YAP/p-YAP ratio was reduced in HPAC and MIA PaCa-2 cell lines and remained unchanged in BxPC-3 cells when cultured in a 3D setting. CTGF and CYR61 gene expression were down-regulated in all PDAC 3D compared to 2D cultures, without any significant effect following actin cytoskeleton inhibition by Cytochalasin B (CyB) treatment. Moreover, LATS1 mRNA, indicating the activation of the Hippo pathway, was not influenced by CyB and differed in all PDAC cell lines having different EMT-related phenotype but a similar pattern of CTGF and CYR61 expression. Although the role of YAP modulation in response to mechanical cues in cancer cells remains to be completely elucidated, our results suggest that cell arrangement and phenotype can determine variable outcomes to mechanical stimuli in PDAC cells. Moreover, it is possible to speculate that YAP and Hippo pathways may act as parallel and not exclusive inputs that, converging at some points, may impact cell behavior. Full article

(This article belongs to the Special Issue The Cytoskeleton: Structural, Functional, and Pathological Aspects)

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16 pages, 4244 KiB

Open AccessArticle

Drosophila Nesprin-1 Isoforms Differentially Contribute to Muscle Function

by Alexandre Rey, Laurent Schaeffer, Bénédicte Durand and Véronique Morel

Cells 2021, 10(11), 3061; https://doi.org/10.3390/cells10113061 - 06 Nov 2021

Cited by 4 | Viewed by 2031

Abstract

Nesprin-1 is a large scaffold protein connecting nuclei to the actin cytoskeleton via its KASH and Calponin Homology domains, respectively. Nesprin-1 disconnection from nuclei results in altered muscle function and myonuclei mispositioning. Furthermore, Nesprin-1 mutations are associated with muscular pathologies such as Emery [...] Read more.

Nesprin-1 is a large scaffold protein connecting nuclei to the actin cytoskeleton via its KASH and Calponin Homology domains, respectively. Nesprin-1 disconnection from nuclei results in altered muscle function and myonuclei mispositioning. Furthermore, Nesprin-1 mutations are associated with muscular pathologies such as Emery Dreifuss muscular dystrophy and arthrogryposis. Nesprin-1 was thus proposed to mainly contribute to muscle function by controlling nuclei position. However, Nesprin-1′s localisation at sarcomere’s Z-discs, its involvement in organelles’ subcellular localization, as well as the description of numerous isoforms presenting different combinations of Calponin Homology (CH) and KASH domains, suggest that the contribution of Nesprin-1 to muscle functions is more complex. Here, we investigate the roles of Nesprin-1/Msp300 isoforms in muscle function and subcellular organisation using Drosophila larvae as a model. Subsets of Msp300 isoform were down-regulated by muscle-specific RNAi expression and muscle global function and morphology were assessed. We show that nuclei anchoring in mature muscle and global muscle function are disconnected functions associated with different Msp300 isoforms. Our work further uncovers a new and unsuspected role of Msp300 in myofibril registration and nuclei peripheral displacement supported by Msp300 CH containing isoforms, a function performed by Desmin in mammals. Full article

(This article belongs to the Special Issue The Cytoskeleton: Structural, Functional, and Pathological Aspects)

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13 pages, 3428 KiB

Open AccessArticle

Mapping the Proximity Interaction Network of STIM1 Reveals New Mechanisms of Cytoskeletal Regulation

by Jesse Gammons, Janith Halpage and Salvatore Mancarella

Cells 2021, 10(10), 2701; https://doi.org/10.3390/cells10102701 - 09 Oct 2021

Cited by 3 | Viewed by 2320

Abstract

Stromal interaction molecule 1 (STIM1) resides primarily in the sarco/endoplasmic reticulum, where it senses intraluminal Ca²⁺ levels and activates Orai channels on the plasma membrane to initiate Ca²⁺ influx. We have previously shown that STIM1 is involved in the dynamic remodeling [...] Read more.

Stromal interaction molecule 1 (STIM1) resides primarily in the sarco/endoplasmic reticulum, where it senses intraluminal Ca²⁺ levels and activates Orai channels on the plasma membrane to initiate Ca²⁺ influx. We have previously shown that STIM1 is involved in the dynamic remodeling of the actin cytoskeleton. However, the downstream effectors of STIM1 that lead to cytoskeletal remodeling are not known. The proximity-labeling technique (BioID) can capture weak and transient protein-protein interactions, including proteins that reside in the close vicinity of the bait, but that may not be direct binders. Hence, in the present study, we investigated the STIM1 interactome using the BioID technique. A promiscuous biotin ligase was fused to the cytoplasmic C-terminus of STIM1 and was stably expressed in a mouse embryonic fibroblast (MEF) cell line. Screening of biotinylated proteins identified several high confidence targets. Here, we report Gelsolin (GSN) as a new member of the STIM1 interactome. GSN is a Ca²⁺-dependent actin-severing protein that promotes actin filament assembly and disassembly. Results were validated using knockdown approaches and immunostaining. We tested our results in neonatal cardiomyocytes where STIM1 overexpression induced altered actin dynamics and cytoskeletal instability. This is the first time that BioID assay was used to investigate the STIM1 interactome. Our work highlights the role of STIM1/GSN in the structure and function of the cytoskeleton. Full article

(This article belongs to the Special Issue The Cytoskeleton: Structural, Functional, and Pathological Aspects)

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29 pages, 9361 KiB

Open AccessArticle

Gelsolin Contributes to the Motility of A375 Melanoma Cells and This Activity Is Mediated by the Fibrous Extracellular Matrix Protein Profile

by Ewa Mazurkiewicz, Aleksandra Makowiecka, Ewa Mrówczyńska, Iryna Kopernyk, Dorota Nowak and Antonina Joanna Mazur

Cells 2021, 10(8), 1848; https://doi.org/10.3390/cells10081848 - 21 Jul 2021

Cited by 8 | Viewed by 3437

Abstract

Skin melanocytes reside on the basement membrane (BM), which is mainly composed of laminin, collagen type IV, and proteoglycans. For melanoma cells, in order to invade into the skin, melanocytes must cross the BM. It has been reported that changes in the composition [...] Read more.

Skin melanocytes reside on the basement membrane (BM), which is mainly composed of laminin, collagen type IV, and proteoglycans. For melanoma cells, in order to invade into the skin, melanocytes must cross the BM. It has been reported that changes in the composition of the BM accompany melanocytes tumorigenesis. Previously, we reported high gelsolin (GSN)—an actin-binding protein—levels in melanoma cell lines and GSN’s importance for migration of A375 cells. Here we investigate whether melanoma cells migrate differently depending on the type of fibrous extracellular matrix protein. We obtained A375 melanoma cells deprived of GSN synthesis and tested their migratory properties on laminin, collagens type I and IV, fibronectin, and Matrigel, which resembles the skin’s BM. We applied confocal and structured illuminated microscopy (SIM), gelatin degradation, and diverse motility assays to assess GSN’s influence on parameters associated with cells’ ability to protrude. We show that GSN is important for melanoma cell migration, predominantly on laminin, which is one of the main components of the skin’s BM. Full article

(This article belongs to the Special Issue The Cytoskeleton: Structural, Functional, and Pathological Aspects)

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22 pages, 10533 KiB

Open AccessArticle

Dual Impact of a Benzimidazole Resistant β-Tubulin on Microtubule Behavior in Fission Yeast

by Mamika Minagawa, Minamo Shirato, Mika Toya and Masamitsu Sato

Cells 2021, 10(5), 1042; https://doi.org/10.3390/cells10051042 - 28 Apr 2021

Cited by 5 | Viewed by 3574

Abstract

The cytoskeleton microtubule consists of polymerized αβ-tubulin dimers and plays essential roles in many cellular events. Reagents that inhibit microtubule behaviors have been developed as antifungal, antiparasitic, and anticancer drugs. Benzimidazole compounds, including thiabendazole (TBZ), carbendazim (MBC), and nocodazole, are prevailing microtubule poisons [...] Read more.

The cytoskeleton microtubule consists of polymerized αβ-tubulin dimers and plays essential roles in many cellular events. Reagents that inhibit microtubule behaviors have been developed as antifungal, antiparasitic, and anticancer drugs. Benzimidazole compounds, including thiabendazole (TBZ), carbendazim (MBC), and nocodazole, are prevailing microtubule poisons that target β-tubulin and inhibit microtubule polymerization. The molecular basis, however, as to how the drug acts on β-tubulin remains controversial. Here, we characterize the S. pombe β-tubulin mutant nda3-TB101, which was previously isolated as a mutant resistance to benzimidazole. The mutation site tyrosine at position 50 is located in the interface of two lateral β-tubulin proteins and at the gate of a putative binging pocket for benzimidazole. Our observation revealed two properties of the mutant tubulin. First, the dynamics of cellular microtubules comprising the mutant β-tubulin were stabilized in the absence of benzimidazole. Second, the mutant protein reduced the affinity to benzimidazole in vitro. We therefore conclude that the mutant β-tubulin Nda3-TB101 exerts a dual effect on microtubule behaviors: the mutant β-tubulin stabilizes microtubules and is insensitive to benzimidazole drugs. This notion fine-tunes the current elusive molecular model regarding binding of benzimidazole to β-tubulin. Full article

(This article belongs to the Special Issue The Cytoskeleton: Structural, Functional, and Pathological Aspects)

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Review

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18 pages, 1419 KiB

Open AccessReview

Neurons: The Interplay between Cytoskeleton, Ion Channels/Transporters and Mitochondria

by Paola Alberti, Sara Semperboni, Guido Cavaletti and Arianna Scuteri

Cells 2022, 11(16), 2499; https://doi.org/10.3390/cells11162499 - 11 Aug 2022

Cited by 10 | Viewed by 2903

Abstract

Neurons are permanent cells whose key feature is information transmission via chemical and electrical signals. Therefore, a finely tuned homeostasis is necessary to maintain function and preserve neuronal lifelong survival. The cytoskeleton, and in particular microtubules, are far from being inert actors in [...] Read more.

Neurons are permanent cells whose key feature is information transmission via chemical and electrical signals. Therefore, a finely tuned homeostasis is necessary to maintain function and preserve neuronal lifelong survival. The cytoskeleton, and in particular microtubules, are far from being inert actors in the maintenance of this complex cellular equilibrium, and they participate in the mobilization of molecular cargos and organelles, thus influencing neuronal migration, neuritis growth and synaptic transmission. Notably, alterations of cytoskeletal dynamics have been linked to alterations of neuronal excitability. In this review, we discuss the characteristics of the neuronal cytoskeleton and provide insights into alterations of this component leading to human diseases, addressing how these might affect excitability/synaptic activity, as well as neuronal functioning. We also provide an overview of the microscopic approaches to visualize and assess the cytoskeleton, with a specific focus on mitochondrial trafficking. Full article

(This article belongs to the Special Issue The Cytoskeleton: Structural, Functional, and Pathological Aspects)

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13 pages, 967 KiB

Open AccessReview

Role of Cytoskeletal Diaphanous-Related Formins in Hearing Loss

by Chiara Chiereghin, Michela Robusto, Valentina Massa, Pierangela Castorina, Umberto Ambrosetti, Rosanna Asselta and Giulia Soldà

Cells 2022, 11(11), 1726; https://doi.org/10.3390/cells11111726 - 24 May 2022

Cited by 4 | Viewed by 2187

Abstract

Hearing relies on the proper functioning of auditory hair cells and on actin-based cytoskeletal structures. Diaphanous-related formins (DRFs) are evolutionarily conserved cytoskeletal proteins that regulate the nucleation of linear unbranched actin filaments. They play key roles during metazoan development, and they seem particularly [...] Read more.

Hearing relies on the proper functioning of auditory hair cells and on actin-based cytoskeletal structures. Diaphanous-related formins (DRFs) are evolutionarily conserved cytoskeletal proteins that regulate the nucleation of linear unbranched actin filaments. They play key roles during metazoan development, and they seem particularly pivotal for the correct physiology of the reproductive and auditory systems. Indeed, in Drosophila melanogaster, a single diaphanous (dia) gene is present, and mutants show sterility and impaired response to sound. Vertebrates, instead, have three orthologs of the diaphanous gene: DIAPH1, DIAPH2, and DIAPH3. In humans, defects in DIAPH1 and DIAPH3 have been associated with different types of hearing loss. In particular, heterozygous mutations in DIAPH1 are responsible for autosomal dominant deafness with or without thrombocytopenia (DFNA1, MIM #124900), whereas regulatory mutations inducing the overexpression of DIAPH3 cause autosomal dominant auditory neuropathy 1 (AUNA1, MIM #609129). Here, we provide an overview of the expression and function of DRFs in normal hearing and deafness. Full article

(This article belongs to the Special Issue The Cytoskeleton: Structural, Functional, and Pathological Aspects)

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14 pages, 1848 KiB

Open AccessReview

Ubiquitin Proteasome System and Microtubules Are Master Regulators of Central and Peripheral Nervous System Axon Degeneration

by Daniele Cartelli, Guido Cavaletti, Giuseppe Lauria and Cristina Meregalli

Cells 2022, 11(8), 1358; https://doi.org/10.3390/cells11081358 - 15 Apr 2022

Cited by 4 | Viewed by 2863

Abstract

Axonal degeneration is an active process that differs from neuronal death, and it is the hallmark of many disorders affecting the central and peripheral nervous system. Starting from the analyses of Wallerian degeneration, the simplest experimental model, here we describe how the long [...] Read more.

Axonal degeneration is an active process that differs from neuronal death, and it is the hallmark of many disorders affecting the central and peripheral nervous system. Starting from the analyses of Wallerian degeneration, the simplest experimental model, here we describe how the long projecting neuronal populations affected in Parkinson’s disease and chemotherapy-induced peripheral neuropathies share commonalities in the mechanisms and molecular players driving the earliest phase of axon degeneration. Indeed, both dopaminergic and sensory neurons are particularly susceptible to alterations of microtubules and axonal transport as well as to dysfunctions of the ubiquitin proteasome system and protein quality control. Finally, we report an updated review on current knowledge of key molecules able to modulate these targets, blocking the on-going axonal degeneration and inducing neuronal regeneration. These molecules might represent good candidates for disease-modifying treatment, which might expand the window of intervention improving patients’ quality of life. Full article

(This article belongs to the Special Issue The Cytoskeleton: Structural, Functional, and Pathological Aspects)

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