Purification, Reconstitution, and Functional Analysis of Connexin Hemichannels
Methods Mol Biol. 2024;2801:1-16. doi: 10.1007/978-1-0716-3842-2_1.ABSTRACTConnexins are the proteins that form the gap junction channels that are essential for cell-to-cell communication. These channels are formed by head-to-head docking of hemichannels (each from one of two adjacent cells). Free "undocked" hemichannels at the plasma membrane are mostly closed, although they are still important under physiological conditions. However, abnormal and sustained increase in hemichannel activity due to connexin mutations or acquired conditions can produce or contribute to cell damage. For example, mutations of Cx26, a connexin ...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Mariana C Fiori Guillermo A Altenberg Source Type: research
Expression and Function of Connexins in Extracellular Vesicles
Methods Mol Biol. 2024;2801:17-28. doi: 10.1007/978-1-0716-3842-2_2.ABSTRACTExtracellular vesicles (EVs) are recognized as major vehicles for exchange of information across distant cells and tissues, which have been extensively explored for diagnosis and therapeutic purposes. The presence of multiple connexin (Cx) proteins has been described in EVs, where they might facilitate EV-cell communication. However, quantitative changes in Cx levels and functional assessment of Cx channels have only been established for Cx43. In present work, we provide a detailed description of the protocols we have optimized to assess the expres...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: T ânia Martins-Marques Henrique Gir ão Source Type: research
Molecular Dynamics Simulation of Permeation Through Connexin Channels
Methods Mol Biol. 2024;2801:45-56. doi: 10.1007/978-1-0716-3842-2_4.ABSTRACTMolecular dynamics (MD) simulations are a collection of computational tools that can be used to trace intermolecular interactions at the sub-nanometer level. They offer possibilities that are often unavailable to experimental methods, making MD an ideal complementary technique for the understanding a plethora of biological processes. Thanks to significant efforts by many groups of developers around the world, setting up and running MD simulations has become progressively simpler. However, simulating ionic permeation through membrane channels still ...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Francesco Zonta Fabio Mammano Sergio Pantano Source Type: research
Spatial and Temporal Localization of Connexins in Cells Using Confocal Microscopy
Methods Mol Biol. 2024;2801:57-74. doi: 10.1007/978-1-0716-3842-2_5.ABSTRACTThe 21-member connexin family found in humans is the building block of both single-membrane spanning channels (hemichannels) and double-membrane spanning intercellular channels. These large-pore channels are dynamic and typically have a short life span of only a few hours. Imaging connexins from the time of synthesis in the endoplasmic reticulum through to their degradation can be challenging given their distinct assembly states and transient residences in many subcellular compartments. Here, we describe how connexins can be effectively imaged on a...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Sergiu A Lucaciu Stephanie E Leighton Dale W Laird Source Type: research
Assessment of Connexin43 Hemichannel Functionality Based on Cytosolic Uptake of Yo-Pro1
Methods Mol Biol. 2024;2801:75-85. doi: 10.1007/978-1-0716-3842-2_6.ABSTRACTConnexin proteins are the building blocks of gap junctions and connexin hemichannels. Both provide a pathway for cellular communication. Gap junctions support intercellular communication mechanisms and regulate homeostasis. In contrast, open connexin hemichannels connect the intracellular compartment and the extracellular environment, and their activation fuels inflammation and cell death. The development of clinically applicable connexin hemichannel blockers for therapeutic purposes is therefore gaining momentum. This chapter describes a well-esta...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Raf Van Campenhout Julen Sanz-Serrano Axelle Cooreman Yannick De Vlaeminck Karine Breckpot Prashant Kadam Andr és Tabernilla Mathieu Vinken Source Type: research
Measurement of Ca < sup > 2+ < /sup > Uptake Through Connexin Hemichannels
Methods Mol Biol. 2024;2801:97-109. doi: 10.1007/978-1-0716-3842-2_8.ABSTRACTIncreasing evidence points to deregulated flux of ionized calcium (Ca2+) mediated by hyperactive mutant connexin (Cx) hemichannels (HCs) as a common gain-of-function etiopathogenetic mechanism for several diseases, ranging from skin disorders to nervous system defects. Furthermore, the opening of nonmutated Cx HCs is associated with an impressive list of widespread diseases including, but not limited to, ischemia/stroke, Alzheimer's disease, and epilepsy. HC inhibitors are attracting a growing attention due to their therapeutic potential for numer...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Chiara Nardin Fabio Mammano Source Type: research
Evaluation of Connexin Hemichannel Activity In Vivo
Methods Mol Biol. 2024;2801:111-124. doi: 10.1007/978-1-0716-3842-2_9.ABSTRACTConnexin hemichannels (Cx HCs) are hexameric structures at the cell plasma membrane, whose function as membrane transport proteins allows for the passive flow of small hydrophilic molecules and ions (≤1 kDa) between the cytosol and the extracellular environment. Activation of Cx HCs is highly dependent on pathological conditions. HC activity provokes changes in the microenvironment, inducing the dissemination of signaling molecules in both an autocrine and paracrine manner. Given the elicitation of a variety of signaling pathways, and assortmen...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Francisca M Acosta Manuel A Riquelme Sumin Gu Jean X Jiang Source Type: research
Enhanced Methodologies for Investigating the Electrophysiological Characteristics of Endogenous Pannexin 1 Intercellular Cell-Cell Channels
Methods Mol Biol. 2024;2801:135-145. doi: 10.1007/978-1-0716-3842-2_11.ABSTRACTGap junctions, pivotal intercellular conduits, serve as communication channels between adjacent cells, playing a critical role in modulating membrane potential distribution across cellular networks. The family of Pannexin (Panx) proteins, in particular Pannexin1 (Panx1), are widely expressed in vertebrate cells and exhibit sequence homology with innexins, the invertebrate gap junction channel constituents. Despite being ubiquitously expressed, detailed functional and pharmacological properties of Panx1 intercellular cell-cell channels require fu...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Nicol ás Palacios-Prado Teresa Vergara Juan C S áez Source Type: research
Generation of Connexin-Expressing Stable Cell Pools
We describe a step-by-step protocol to produce the LV particle by transient transfection and the direct use of the harvested LV stock to generate stable cell pools. We further present step-by-step immunolabeling protocols to characterize the transgene protein expression by confocal microscopy using an antibody that targets an extracellular domain epitope of Cx32 in living cells, and in fixed permeabilized cells using high affinity anti-Cx32 antibodies. Using common molecular biology laboratory techniques, this protocol can be adapted to generate stable pools expressing any transgene of interest, for both in vitro and in vi...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Abraham Tettey-Matey Chiara Di Pietro Viola Donati Fabio Mammano Daniela Marazziti Source Type: research
A Protocol for the Automated Assessment of Cutaneous Pathology in a Mouse Model of Hemichannel Dysfunction
Methods Mol Biol. 2024;2801:177-187. doi: 10.1007/978-1-0716-3842-2_13.ABSTRACTIn this chapter, we provide detailed instructions to perform quantitative reflectance imaging in a mouse model of a rare epidermal disorder caused by hyperactive connexin 26 hemichannels. Reflectance imaging is a versatile and powerful tool in dermatology, offering noninvasive, high-resolution insights into skin pathology, which is essential for both clinical practice and research. This approach offers several advantages and applications. Unlike traditional biopsy, reflectance imaging is noninvasive, allowing for real-time, in vivo examination o...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Chiara Peres Fabio Mammano Source Type: research
Measuring Connexin Hemichannel Opening in Response to an InsP3-Mediated Cytosolic Ca < sup > 2+ < /sup > Increase
Methods Mol Biol. 2024;2801:189-197. doi: 10.1007/978-1-0716-3842-2_14.ABSTRACTThe opening of connexin hemichannels (HCs) expressed at the plasma membrane of mammalian cells is regulated by a number of physiological parameters, including extracellular and intracellular Ca2+ ions. Submicromolar variations of the cytosolic Ca2+ concentration ([Ca2+]c) are per se sufficient to trigger extracellular bursts of messenger molecules through connexin HCs, thus mediating paracrine signaling. In this chapter, we present a quantitative method to measure the opening dynamics of connexin HCs expressed in a single HeLa cell upon stimulat...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Erva Bayraktar Mario Bortolozzi Source Type: research
Determination of anti-cancer effects of Nigella sativa seed oil on MCF7 breast and AGS gastric cancer cells
CONCLUSIONS: Our findings indicate that N. sativa oil has a dose-dependent effect on cytotoxicity and the expression of apoptotic, heat shock proteins, and matrix metalloproteinases genes in breast and gastric cancer.PMID:38578469 | DOI:10.1007/s11033-024-09453-1 (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: İrfan Çınar Bet ül Gıdık Ebubekir Dirican Source Type: research
Harnessing potential of avian eggshell membrane derived collagen hydrolysate for bone tissue regeneration
CONCLUSIONS: The maximum ratio of Col-h/PVA that produced well defined hydrogels was 50:50. Though all the hydrogel matrices alluded towards their competitive attributes and applicability towards restorative bone repair, the hydrogel with 40:60 ratios showed better mechanical strength and cell proliferation than its counterparts. The prominent E. coli growth inhibition by the hydrogel matrices was also observed, along with excellent biocompatibility with MG-63 osteoblasts. The findings indicate strongly the promising application of avian eggshell-derived Col-h in supporting bone regeneration.PMID:38578512 | DOI:10.1007/s11...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Aakriti Aggarwal Debasish Nath Asish Pal Mahesh Kumar Sah Source Type: research
Characterization of Bitter Taste Receptor-Dependent Autophagy in Oral Epithelial Cells
Methods Mol Biol. 2024 Apr 6. doi: 10.1007/7651_2024_531. Online ahead of print.ABSTRACTMicrobial dysbiosis is an important trigger in the development of oral diseases. Oral keratinocytes or gingival epithelial cells (GECs) offer protection against various microbial insults. Recent studies suggest that GECs expressed higher level of bitter taste receptor 14 (T2R14) compared to other taste receptors and toll-like receptors and act as innate immune sentinels. Macroautophagy or autophagy is a cellular conserved process involved in the regulation of host innate immune responses against microbial infection. Here, we describe a ...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Nisha Singh Saeid Ghavami Prashen Chelikani Source Type: research
Regeneration of Volumetric Muscle Loss Using MSCs Encapsulated in PRP-Derived Fibrin Microbeads
Methods Mol Biol. 2024 Apr 6. doi: 10.1007/7651_2024_533. Online ahead of print.ABSTRACTVolumetric muscle loss (VML) is one of the major types of soft tissue injury frequently encountered worldwide. In case of VML, the endogenous regenerative capacity of the skeletal muscle tissue is usually not sufficient for complete healing of the damaged area resulting in permanent functional musculoskeletal impairment. Therefore, the development of new tissue engineering approaches that will enable functional skeletal muscle regeneration by overcoming the limitations of current clinical treatments for VML injuries has become a critica...
Source: Mol Biol Cell - April 5, 2024 Category: Molecular Biology Authors: Şükran Şeker Özge Lalegül-Ülker Ay şe Eser Elçin Ya şar Murat Elçin Source Type: research
