Dendra2-tagged proteins can therefore be used for tracking protein dynamics, remodelling and turnover using super resolution microscopy.Ī549 lung adenocarcinoma cells (ATCC ® CCL-185™) were maintained at 37 ☌, 5% CO 2 in high glucose (4.5 g/L) Dulbecco’s Modified Eagle Media with 2 mM l-glutamine (Sigma-Aldrich, St. Dendra2 emits green fluorescence under blue light however, exposure to short wavelength light photo converts the protein from green to red. We selected LMβ1 tagged with the photoconvertible protein Dendra2. Here we aimed to establish an in vitro model to study human LM dynamics. Advances in CRISPR-Cas9 genome editing present an opportunity to directly tag endogenous human LM genes for stable expression. However, these constructs are at the adenoviral packaging limit and allow only transient expression. In human cells, adenoviral-mediated expression of the two smallest LMs, LMβ3 and LMγ2, with C-terminal fluorescent tags has also been performed. elegans described genetically tagging the C-terminus of the worm LMβ chain ortholog with fluorescent proteins, allowing in vivo observation of BM turnover and remodelling.
#Snapgene confocal microscopy series
Recently, a series of elegant studies in C. Despite extensive investigation, surprisingly little is known regarding the mechanisms and dynamics of LM deposition and remodelling, in part owing to lack of appropriate tools for viewing these nanoscale process in live conditions. In the α chains, the LCC is followed by five globular domains which contain the major cell-surface receptor binding sites while at the amino terminus of a subset of LMs are LN domains involved in LM network assembly (Fig. LM heterotrimers assemble intracellularly via an α-helical laminin coiled coil (LCC) domain in each chain. Each LM is an αβγ heterotrimer comprising one of five α chains, one of three β chains and one of three γ chains, each derived from a distinct gene. Laminin (LM) are core components of all basement membranes (BMs) and are essential for tissue function by providing a substrates for cell attachment and migration, a barrier to tumour invasion, and in regulating signalling pathways. Together, these data suggest that, in humans, addition of C-terminal Dendra2 tag to LMβ1 inhibits LM secretion, and is not a viable approach for use in animal models. Moreover, the edited cells displayed reduced proliferation rates. Despite expression of the tagged protein within cells, no detectable LMβ1-Dendra2 protein was deposited to the extracellular matrices or conditioned media of edited cells. ResultsĬRISPR-Cas9 was used to introduce the Dendra2 sequence at the C-terminus of LMβ1 in the human lung adenocarcinoma cell line A549. Here we used genome editing to establish a similar system in a mammalian cell line as proof of concept for future mammalian models. elegans ortholog of human the LMβ1 chain was labelled at the C-terminus with the photoconvertible fluorophore Dendra2. However, surprisingly little is known about the mechanisms of turnover and remodelling of LM networks due to lack of appropriate tools to study these processes at the necessary resolution. The laminins (LM) are a family of basement membranes glycoproteins with essential roles in supporting epithelia, endothelia, nerves and muscle adhesion, and in regulating a range of processes including cell migration, stem cell maintenance and differentiation.
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