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Optogenetic dissection of mitotic spindle positioning in vivo

Lars-Eric Fielmich, Ruben Schmidt, Daniel J Dickinson, Bob Goldstein, Anna Akhmanova, Sander van den Heuvel

Preprint posted on May 14, 2018 https://www.biorxiv.org/content/early/2018/05/13/319772

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

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.

A maximum intensity projection of a z-stack through an L4 larva expressing PH::eGFP::LOV. It is under the control of an ubiquitous promoter and thus visualizes all the membranes in the worm. For instance, one of the gonads is nicely visible (it is not silenced in the germline). This strain was central to the study. Image and caption provided by authors.

 

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

 

Posted on: 5th August 2018

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  • Author's response

    Lars-Eric Fielmich and Ruben Schmidt shared

    We are excited that our work is being noted and appreciated by the scientific community. We hope that this highlight on preLight will improve the reach of our findings and methods. In addition to the elaborate highlight of our method, we would like to explain why this optogenetic method was essential to reach our conclusion. For our research question, we wanted to investigate the functions of the conserved LIN-5NuMA protein complex in C. elegans mitotic spindle positioning. The LIN-5 complex recruits the motor protein dynein and constitutes a cortical force generator that pulls on the astral microtubules of the spindle. Because all members of the LIN-5 complex are also part of a structural anchor for dynein, individual protein functions had not been ascribed. Using the optogenetic heterodimerization of ePDZ and LOV protein tags, we were able to reconstitute cortical force generators with different protein compositions, while maintaining the anchor structure. This would not have been possible with more traditional techniques. Doing so, we uncovered an essential role of LIN-5 as activator of the dynein motor protein. This is contrary to previous research that had concluded that the LIN-5 complex merely served as a dynein anchor.

    In answer to the questions posed by the preLight team:

    • How widely applicable are these gene silencing mechanisms and the methods to overcome them?

    Germline gene silencing is a conserved mechanism for transposon silencing and regulation of germline gene expression. It has also been described in plants, insects, and vertebrates including mammals. It has impeded the research progress in quite a few studies, also outside the C. elegans field, and multiple methods to prevent it have been reported. Methods that depend on specific sequences or transgenesis protocols may be species specific. Since our method provides a means to rationally optimize sequences based on RNAseq data, it should be applicable to work in other organisms as well. As an alternative, when an organism’s piRNA are characterized, transgenes can be adjusted to lack piRNA target sites.

    • Is a similar approach being tried in another model system using an available RNAseq dataset?

    There are, to our knowledge, no similar approaches yet in other model systems.

    • Can other optogenetic methods such as the phytochrome or cryptochrome systems, be now easily adapted in the roundworm?

    It will be interesting to test other optogenetic methods, in particular phytochrome systems that allow inducible protein association as well as dissociation. Our germline-optimization method not only has helped us to express epdz and lov sequences, but also the expression of the cre recombinase in the germline (this study). This method, and recently developed alternative strategies, appear to efficiently overcome germline silencing in C. elegans and should apply to any transgene sequences, including other optogenetic methods.

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