Optogenetic dissection of mitotic spindle positioning in vivo
Posted on: 5 August 2018
Preprint posted on 14 May 2018
Article now published in eLife at http://dx.doi.org/10.7554/elife.38198
Overcoming severe technical roadblocks, the beautiful and powerful model system of the early C. elegans embryo is now amenable to optogenetic manipulation.
Selected by Angika Basant
Categories: cell biology, developmental biology
Background: This bioRxiv study investigates positioning mechanisms of the mitotic spindle in the early C. elegans embryo. In the course of addressing their research question, the authors also establish optogenetics in this model system, an exciting technical advance that I would like to highlight.
The nematode embryo is an important, genetically tractable model system in cell biology that is highly conducive for live imaging. Important aspects of cell polarity (1), meiosis (2), phase separation (3) and cytokinesis (4) have been elucidated using the nematode zygote. Desired genetic mutations, tagged fluorescent proteins, CRISPR or RNAi-based knock-downs are readily generated/available in this organism.
Optogenetics is a technique in biology where light sensitive proteins are expressed in cells or tissues to manipulate the activity or localisation of molecules of interest. Its scope in cell biology is gradually moving beyond proof-of-principle experiments, to provide mechanistic insight into key cellular processes (5, 6). However, stable expression of “foreign” DNA sequences of light-responsive genes (typically adapted from plants) in animals can be challenging; transient expression in cultured cells has been more successful. In C. elegans, optogenetics approaches have been applied to somatic cells (7) but germline cells, including the early fertilized egg, demonstrate strong gene silencing responses towards integrated transgenes.
This study aimed to use the TULIPs optogenetic system (10) to recruit proteins involved in mitotic spindle positioning to the plasma membrane, and investigate their precise functions. TULIPs utilises a light-responsive LOV2 domain, the C terminus of which contains a short peptide designed to bind an engineered PDZ domain (ePDZ) upon illumination with blue light. By linking it with an appropriate interacting domain, LOV2 can be localised anywhere in the cell, such as the plasma membrane in this study. ePDZ can be fused to a protein of interest to allow its light-dependent, subcellular recruitment to a desired site.
Key findings: To express these optogenetic components in the one-cell C. elegans zygote, the authors developed an algorithm to optimise exogenous sequences, by incorporating characteristics of endogenous genes. By mining an RNAseq dataset of genes expressed in the early embryo, they generated a list of 12-mer sequences and assigned each a score based on how frequently they were found, and the expression levels of the genes they were found in. Next, based on this list, their algorithm chose a “high-scoring” exonal gene sequence for the desired amino acid chain (such as GFP-LOV2 or mCherry-ePDZ). This germline optimization dramatically improved the expression levels of genes notorious for being silenced, such as gfp::cdk-1. However, the genome-integrated lines thus generated lost expression of desired genes after a certain number of generations.
It has recently been reported that endogenous genes contain Periodic A/T Clusters (PATCs) in their introns that help evade silencing (8). Therefore, to express TULIPs components in the germline, in addition to optimizing the exon sequence of exogenous genes, PATC sequences were introduced into intronic regions. This resulted in stable expression of both LOV2 and ePDZ fused proteins in the C. elegans germline. In the absence of blue light, ePDZ was shown to be exclusively localised to the cytosol, but could be robustly and reversibly recruited to desired regions of the plasma membrane in the first cell cycle of the zygote.
What I like about this preprint: This study builds on our understanding of regulation of gene expression, and applies it to create an important optogenetic tool for many fields of biology, including cell, developmental and neurobiology. Expression of foreign sequences in model organisms has always required standardization, for example codon optimisation of gene sequences is a common practice. This study develops an innovative, computational approach to predictably assemble a coding sequence for superior gene expression, that may be generalisable to other contexts. Further, based on the recently revealed importance of non-coding regions in resisting gene silencing, the intronic regions of optogenetic components were optimised for robust and stable expression across generations.
Future directions and questions for the authors: How widely applicable are these gene silencing mechanisms and the methods to overcome them? Is a similar approach being tried in another model system using an available RNAseq dataset? And can other optogenetic methods such as the phytochrome or cryptochrome systems, be now easily adapted in the roundworm?
(1) Munro et al., Dev Cell 2004
(2) Bhalla and Dernburg, Science 2005
(3) Smith et al., eLife 2016
(4) Jantsch-Plunger et al., JCB 2000
(5) Wagner et al., JCB 2016
(6) Ramachandran et al., eLife 2018
(7) Harterink et al., Curr Biol 2016
(8) Frøkjær-Jensen et al., Cell 2016
(9) Zhang et al., Science 2018
(10) Strickland et al., Nat Meth 2010
doi: https://doi.org/10.1242/prelights.4111
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