Cytochalasin G

Cortical actin plays a key role in cell movement and division, but has also been implicated in the organisation of cell surface receptors such as G protein-coupled receptors. The actin mesh proximal to the inner membrane forms small fenced regions, or 'corrals', in which receptors can be constrained. Quantification of the actin mesh at the nanoscale has largely been attempted in single molecule datasets and electron micrographs. This work describes the development and validation of workflows for analysis of super resolved fixed cortical actin images obtained by Super Resolved Radial Fluctuations (SRRF), Structured Illumination Microscopy (3D-SIM) and Expansion Microscopy (ExM). SRRF analysis was used to show a significant increase in corral area when treating cells with the actin disrupting agent cytochalasin D (increase of 0.31 µm2 ± 0.04 SEM), and ExM analysis allowed for the quantitation of actin filament densities. Thus, this work allows complex actin networks to be quantified from super-resolved images and is amenable to both fixed and live cell imaging.

Phenochalasin A, a unique phenol-containing cytochalasin produced by the marine-derived fungus Phomopsis sp. FT-0211, was originally discovered in a cell morphological assay of observing the inhibition of lipid droplet formation in mouse peritoneal macrophages. To investigate the mode of action and binding proteins, phenochalasin A was radio-labeled by 125I. Iodinated phenochalasin A exhibited the same biological activity as phenochalasin A. [125I]Phenochalasin A was found to be associated with an approximately 40 kDa protein, which was identified as G-actin. Furthermore, detail analyses of F-actin formation in Chinese hamster ovary cells (CHO-K1 cells) indicated that phenochalasin A (2 µM) caused elimination of F-actin formation on the apical site of the cells, suggesting that actin-oriented specific function(s) in cytoskeletal processes are affected by phenochalasin A.

Although three-dimensional (3D) co-culture of gingival keratinocytes and fibroblasts-populated collagen gel can mimic 3D structure of in vivo tissue, the uncontrolled contraction of collagen gel restricts its application in clinical and experimental practices. We here established a stable 3D gingival tissue equivalent (GTE) using hTERT-immortalized gingival fibroblasts (hGFBs)-populated collagen gel directly crosslinked with genipin/cytochalasin D and seeding hTERT-immortalized gingival keratinocytes (TIGKs) on the upper surface for a 2-week air-liquid interface co-culture. MTT assay was used to measure the cell viability of GTEs. GTE size was monitored following culture period, and the contraction was analyzed. Immunohistochemical assay was used to analyze GTE structure. qRT-PCR was conducted to examine the mRNA expression of keratinocyte-specific genes. Fifty µM genipin (G50) or combination (G + C) of G50 and 100 nM cytochalasin D significantly inhibited GTE contraction. Additionally, a higher cell viability appeared in GTEs crosslinked with G50 or G + C. GTEs crosslinked with genipin/cytochalasin D showed a distinct multilayered stratified epithelium that expressed keratinocyte-specific genes similar to native gingiva. Collagen directly crosslinked with G50 or G + C significantly reduced GTE contraction without damaging the epithelium. In summary, the TIGKs and hGFBs can successfully form organotypic multilayered cultures, which can be a valuable tool in the research regarding periodontal disease as well as oral mucosa disease. We conclude that genipin is a promising crosslinker with the ability to reduce collagen contraction while maintaining normal cell function in collagen-based oral tissue engineering.