How to Train a Cell–Cutting-Edge Molecular Tools

Abstract

In biological systems, the formation of molecular complexes is the currency for all cellular processes. Traditionally, functional experimentation was targeted to single molecular players in order to understand its effects in a cell or animal phenotype. In the last few years, we have been experiencing rapid progress in the development of ground-breaking molecular biology tools that affect the metabolic, structural, morphological, and (epi)genetic instructions of cells by chemical, optical (optogenetic) and mechanical inputs. Such precise dissection of cellular processes is not only essential for a better understanding of biological systems, but will also allow us to better diagnose and fix common dysfunctions. Here, we present several of these emerging and innovative techniques by providing the reader with elegant examples on how these tools have been implemented in cells, and, in some cases, organisms, to unravel molecular processes in minute detail. We also discuss their advantages and disadvantages with particular focus on their translation to multicellular organisms for in vivo spatiotemporal regulation. We envision that further developments of these tools will not only help solve the processes of life, but will give rise to novel clinical and industrial applications.

Advances in understanding nociception and neuropathic pain

“…Two further techniques recently utilised to further understanding of nociceptor function and circuitry are optogenetics and chemogenetics. Optogenetics involves inducing neuronal expression of light-activated membrane proteins whose activation can enable either depolarisation (e.g., channelrhodopsin-2, ChR2) or hyperpolarisation (e.g., halorhodopsin) to directly switch-on or switch-off neurones, respectively, as well as switching on of G protein-coupled receptor (GPCR) signalling cascades [ 33 ]. By contrast, chemogenetics entails, among other strategies, designer receptors exclusively activated by designer drugs (DREADDs), which are modified GPCRs no longer activated by their endogenous ligand acetylcholine, but instead the biologically inert substance clozapine- N -oxide (CNO, although recent work suggests that clozapine rather than CNO is the receptor ligand [ 34 ]); differently G protein-coupled DREADDs allow for activation (G s or G q ) or inhibition (G i ) of neuronal function [ 35 ].…”

Optogenetically controlled protein kinases for regulation of cellular signaling

“…However, this approach does not allow the regulation of such parameters as the duration of the protein kinase signaling or the localization of the activated protein kinases. These parameters can be varied with the help of such methods as the usage of microfluidic devices for temporarily defined growth factor delivery into culture medium 5 , antagonistic reversal of the original signal with the help of different combinations of chemically induced dimerizers (CIDs) 6 and immobilizing patterns of growth factors on fibrin substrates to achieve spatially directed guidance of cell migration 7 .…”

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

“…8D,E) in the dorsal compartment of the discs using apterous-GAL4 (ap-GAL4) resulted in a clear Development • Accepted manuscript reduction of Tkv levels in dorsal cells. This reduction in receptor levels was accompanied by a substantial loss of pMad and Spalt (Sal) expression, a BMP/pMadtarget gene (de Celis and Barrio, 2009) in the same compartment ( Fig. 8F-K).…”

“…Alternatively, chemically regulated modules can also be fused to POIs such that some of their functions (half-life, localization, etc.) can be manipulated ( Banaszynski et al, 2006 ; Bonger et al, 2011 ; Chung et al, 2015 ; Czapiński et al, 2017 ; Natsume and Kanemaki, 2017 ; Natsume et al, 2016 ).…”