My university has reopened and being reunited with the lab bench after months have reminded me again how much I love what I do for a living. Of course I enjoy the entire process of research from bench to publication, troubleshooting experiments and brainstorming ideas to presenting research stories. However, hands-on working is something I relish the most. It has a tangible and stimulatory effect on my experimental thought process and in general how I perceive and approach scientific questions.

Staying home for more than three months has been the largest break in my life since kindergarten and has affected my mental wellbeing. I had moved to Boston as a postdoc to study how forces shape tissue patterning and morphogenesis in zebrafish, after finishing PhD in single-molecule plant development in Tübingen, Germany. However, within two months of my arrival in Boston, Harvard stopped all the non-essential research due to soaring coronavirus pandemic.

Living pretty much alone in house arrest in a new city away from my partner and family has been extremely stressful and emotionally difficult. The new lab has been very supportive and connected throughout but the isolation – paired with the rejection from the postdoctoral fellowship I applied for – has given me an imposter syndrome. I felt the sadness growing, my moods shifting, confidence losing, and fear building in me.  For example, during scientific zoom meetings with the lab and the department, I was not able to communicate my thoughts and ideas properly because being out of the lab and staying in isolation was toying with my state of mind. Of course, afterwards I then dealt with the guilt of it. The murder of George Floyd further added frustration and anger about the world we live in and how we treat each other. I could feel and acknowledge my worsening mental health. Therefore, I constantly talked about my feelings with my partner and my best friend who have been incredibly supportive irrespective of our separation by the continents. Although communication helps, I felt I still need tangible positivity to pull me out of the mental black hole. So, I have been taking time to reflect on positive things. For example, I have been excitedly rethinking about the scientific problems I am interested in and ways to approach them differently. And besides science, reading literary and historical fiction and baking cakes and pastries. I am sure I am not alone in experiencing such mental instability in this time. Each one of us is experiencing stress and anxiety in our own ways. What is most important is to acknowledge the symptoms and seek the help and support we need.

When I heard the university was reopening and we could go back to the lab, I was thrilled. This is exactly the positivity I think I have been longing for. The flipside to this positivity is that I was nervous about going back. I joined a zebrafish developmental and systems biology lab and had learned how to do crossings and microinjections in zebrafish right before the shutdown. Obviously, I have been anxious that I had forgotten the newly learned methods and whether I would be able to find reagents and consumables in the lab.

Now that I am in the lab again, of course, it is all the same as I knew it, physically. Except that the labs, corridors, hallways, bathrooms, meeting points, and common areas are labelled with safety distance reminders and are awfully silent and empty. There are no social hours or physical meetings. It feels like working late over Christmas. Even fish in the lab probably miss the hustle-bustle of colleagues. I, on the other hand, am delighted to be back in the lab and to see a few familiar masked faces around. Foremost, it is getting me back into the workday routine and breaking the monotony of alone and ‘relaxed’ working from home. More importantly, I am thrilled to be working on the bench again. I think there is an eternal joy that we wet-lab scientists find in bench work. To get back into the familiar working spirit, I resumed by molecular cloning experiments, which I am most comfortable with. Seeing bacterial colonies on the agar plate the day after bacterial transformation have already started to diminish my feelings of being an imposter. Turns out, I can still cross zebrafish and pull needles for microinjections. I am starting to find purpose again, after all work is the reason I moved to the US.

Although we are working in spatially and temporally coordinated lab shifts, the motivation to be in the lab and conduct experiments is more than enough for me to be feeling mentally healthy again. I understand it is not the ideal situation for any of us. But neither is the uncertainty of normality. I think by embracing little steps towards normality, we can overcome the mental hurdles posed by the pandemic. I realise my mental clock has started to shift towards normal: being back at the bench is definitely the sweeter side of our bitter-sweet labcoming experience.

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Ziheng Liu and Ying Yang

The asymmetry in cell morphology and the asymmetric distribution of intracellular organelles, proteins, nucleic acids and other components are the hallmarks of cellular polarity, possessed by most, if not all, cells. Polarity plays important roles in cell differentiation and function, and its dysregulation is intimately related to developmental defects, tumor formation and cancer metastasis. The commonality of cell polarization events is that some polarity-regulating proteins (such as the Par3/Par6/aPKC complex, or the Frizzled/Dishevelled/Diego complex) are specifically recruited to the designated subcellular area and form local aggregates, attaching to the inner surface of the plasma membrane. These protein aggregates are dynamic and assemble and disassemble quickly in response to cellular signals [1-3]. How these protein complexes can achieve polarized concentration on the premise of open contact with the cytoplasm while maintaining a high degree of dynamics has been an open question.

In recent years, the “liquid-liquid phase separation” (LLPS) of biomacromolecules has emerged as an important mechanism underlying the formation of a variety of membraneless structures within cells [4,5]. The aggregates of polarity proteins share some similar characteristics with these membraneless structures, such as high condensation and dynamic equilibrium with proteins in cytoplasm.

We showed previously that during the asymmetric divisions of Drosophila larval neural stem cells (or neuroblasts), cell fate determinants including Numb and Pon undergo LLPS. Multivalent interactions lead to the polarized enrichment of the Numb/Pon complex on the basal cortex of dividing neuroblasts, which regulates the differentiation of the ganglion mother cell (GMC), the differentiating daughter of the neuroblast, upon cell cycle exit [6].

On May 8, 2020, we published a research work titled “Par complex cluster formation mediated by phase separation” online in Nature Communications [7]. The paper provides evidence showing that the apical Par protein complex is also assembled via a LLPS mechanism, thereby regulating the establishment of the neuroblast apical-basal polarity and consequently the distinct cell fate of neuroblast daughters.

One of the best-studied polarity complexes, the highly conserved Par complex is composed of Par3 (Bazooka in Drosophila), Par6 and aPKC, which can interact with each other to form a polarity core and further recruit other polarity proteins. During the interphase of neuroblasts, these polarity proteins are distributed throughout the cytoplasm. Upon mitotic entry, Par3, Par6 and aPKC gradually accumulate underneath the apical cortex and form a multiprotein-containing apical complex, which directs the basal enrichment of those cell fate determinants including the Mira/Pros/Brat/pros mRNA complex and the Pon/Numb complex, via aPKC-mediated phosphorylation events. Upon the completion of mitotic cycle, the Par3/Par6/aPKC complex remains in the neuroblast daughter and the complex is dissembled, whilst those cell fate determinants are preferentially segregated into the GMC daughter to promote its differentiation. In this latest work, we showed upon mitotic entry Par3 (or Baz) forms apically-localized liquid droplets, which is mediated by its N-terminal domain (NTD) harboring oligomerization activity. These liquid droplets subsequently fuse and assemble into a highly concentrated cluster around the apical pole, forming the condensates underneath the apical cortex. Par6 is recruited to the Par3 condensate through the specific binding of its C-terminal motif to PDZ3 domain of Par3. Interestingly, the incorporation of Par6 into Par3 condensates greatly promotes the LLPS of Par3 via multivalent interactions.

As the only known kinase in the complex, aPKC can also be recruited and enriched in the Par3/Par6 condensates, but aPKC in the droplets is surprisingly in an inactive state. Interestingly, activated aPKC can phosphorylate Par3 and promote dissolve of the Par3/Par6 aggregates. Based on these observations, we speculate that the formation of the Par condensed droplets may provide an effective platform to recruit and concentrate the limited aPKC from the cytoplasm to the apical sub-membrane area, where aPKC can be activated by cell cycle regulatory factors (such as Cdc42) upon mitotic entry, and subsequently phosphorylate Par3, which eventually leads to the dissembly of the Par protein aggregates. Meanwhile, the activated aPKC also mediates the basal segregation of the fate determinants through its kinase activity. Interfering with the formation of Par3/Par6 LLPS will disrupt the establishment of the apical-basal polarity during the asymmetric divisions of Drosophila neuroblasts, leading to the defects in the development of neuronal lineages (Figure 1). This study, together with our previous work, shows that the LLPS, mediated by the multivalent interactions among these polarity proteins, plays important roles during the establishment of Drosophila neuroblast polarity.

Although LLPS provides a new perspective for the formation of a large number of membraneless structures in cells and the selective enrichment and separation of cellular components in various physiological processes, whether it regulates these biological processes under physiological conditions has always been the focus of debate. One main reason is that LLPS is largely dependent on the protein concentration, and the concentration of the target protein used in cell overexpression systems and in vitro experiments is often higher than that under the physiological conditions.

To test this, we established GFP knock-in Drosophila strains expressing GFP-tagged Baz under its endogenous regulatory elements, and explored the role of LLPS (of Baz) in the establishment of neuroblast polarity at an endogenous setting. In sharp contrast to the overexpression assay results showing that Baz ΔNTD (deleting the N-terminal domain, thus compromising LLPS ability) lost its apical condensation in both WT and baz mutant backgrounds, the knock-in mutant Baz ΔNTD surprisingly exhibited obvious apical condensation (crescent formation). However, it also exhibited significant cytoplasmic diffusion, which likely explains the observed smaller brain phenotype. More importantly, replacing the Baz NTD domain with a low complexity domain (LCD)-containing fragment of FUS, which has no secondary structure but can phase separate, also significantly rescued brain development abnormalities caused by NTD deletion (Figure 1). These results provide strong support for the hypothesis that the polarized distribution and establishment of the Par complex is mediated by LLPS, and also points out the importance of protein concentration when assessing the LLPS for protein of interest. It further suggests that the results obtained form in vitro or over-expression study need to be interpreted with caution.

In summary, our study reveals that the LLPS mediated by the multivalent interactions among the polarity proteins Par3, Par6 and aPKC promotes their condensation beneath the apical membrane, and the rapid response of the phase-separated condensates to cellular signals also ensures a high dynamics in the assembly and disassembly of cellular polarity. Since most polarity complexes contain multidomain proteins able to form condensed puncta, and these puncta are in dynamic equilibrium with proteins in cytoplasm, the LLPS proposed in this study may be a general mechanism for the establishment of cellular polarity.

Figure 1. Phase separation of Par complex regulates the polarity establishment of neuroblast. Figure 9 in Liu, et al. Nature Communications 2020.

Fudan University doctoral student Liu Ziheng, master student Gu Aihong, and National University of Singapore Dr. Yang Ying are co-first authors. Fudan University PI Wen Wenyu and Singapore National University Professor Cai Yu are co-corresponding authors.

1. Rodriguez-Boulan, E. & Macara, I.G. Organization and execution of the epithelial polarity programme. Nat Rev Mol Cell Biol 15, 225-42 (2014).
2. Knoblich, J.A. Asymmetric cell division: recent developments and their implications for tumour biology. Nat Rev Mol Cell Biol 11, 849-60 (2010).
3. Yang, Y. & Mlodzik, M. Wnt-Frizzled/planar cell polarity signaling: cellular orientation by facing the wind (Wnt). Annu Rev Cell Dev Biol 31, 623-46 (2015).
4. Shin, Y. & Brangwynne, C.P. Liquid phase condensation in cell physiology and disease. Science 357(2017).
5. Banani, S.F., Lee, H.O., Hyman, A.A. & Rosen, M.K. Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol 18, 285-298 (2017).
6. Shan, Z. et al. Basal condensation of Numb and Pon complex via phase transition during Drosophila neuroblast asymmetric division. Nature Communications 9, 737 (2018).
7. Liu, Z. et al. Par complex cluster formation mediated by phase separation. Nature Communications, 11(1) (2020).

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In early June, as Black Lives Matter protests were gathering momentum across the globe, a group of publishers were brought together (virtually of course) by the Royal Society of Chemistry (RSC) to discuss equality, diversity and inclusion (EDI) in academic publishing activities. While the timing of this workshop was completely coincidental, the experiences shared by Black researchers through, for example, #BlackintheIvory on Twitter lent a sense of urgency to our meeting. Many publishers, The Company of Biologists included, had already begun to think about disparities and biases in publishing, particularly in terms of gender imbalance (as discussed in our recent editorial), but we all recognise that there is much more we need to do.

What came out of that workshop was a Joint Statement committing to action – to better understand our research communities, better reflect that diversity, and to set and share policies to improve EDI practices across multiple publishers. In the video below, I spoke to our Science Communications Officer, Annabel, about the joint statement and the Company’s plans for the future.

We are very much at the beginning of our EDI journey, but we’re looking forward to working with other publishers to come up with concrete actions we can take. And we’re excited about the opportunities that the Node Network has to help diversify our conferences, departmental seminars and other activities – particularly in the virtual world, geography is no boundary to invitations to speak! If you haven’t already signed up for, or looked at, the Node Network, we encourage you to do so.

And, as always, we’re keen to hear from the community about what we should be doing from an EDI perspective, so please do get in touch!

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This Editorial by Aidan Maartens, Katherine Brown and James Briscoe was published yesterday in Development. Don’t forget that we’re celebrating 10 years with a free online networking event on July 29 – more details here.

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An engineer position in molecular and cellular biology is available starting in September 2020 in the group of Pierre-François Lenne and in collaboration at the Developmental Biology Institute of Marseille (IBDM), France. The initial appointment will be made for 1 year, with a possible extension to up to 3 years.

We are seeking a highly-motivated candidate, who will support an interdisciplinary project on cell dynamics and tissue morphogenesis.

The main tasks of the engineer will be the preparation of cell aggregates from mouse embryonic stem cells (“embryonic organoids”), and the development of cell lines expressing fluorescent reporter proteins to monitor gene expression dynamics through imaging. Specifically, the job requires expert knowledge in cell biology (cell cultures, immuno-staining), imaging (e.g. confocal microscopy) and molecular biology (cloning of large DNA fragments, routine and advanced PCR technology). Expertise in stem- cell biology is desirable.

The working language in the laboratory is English. Candidates are expected to be fluent in English.

A letter of motivation, a CV and the names of two referees should be sent to Pierre-François Lenne

Annoucement in pdf

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FocalPlane is a new microscopy community site hosted by Journal of Cell Science (JCS), Development’s sister journal, and like the Node funded by our not-for-profit publisher, The Company of Biologists. Launched yesterday, it encompasses all fields in the biological sciences where they meet microscopy.

Here the FocalPlane team introduce the site:

From conversations the JCS editorial team had with the microscopy community, it was clear there was the need for a trusted, curated and centralised online meeting place to connect people, products, resources and information relating to microscopy. The idea for FocalPlane was born.

The ability to tackle ever-more-refined biological questions is improving as microscopy and image analysis become increasingly more complex and sophisticated. However, this has made it more and more difficult for non-experts to access user-friendly resources or tools tailored to their questions. Thus, there is a need for a platform for both microscope/software developers and researchers to exchange ideas and information to help the field develop and progress.

To encourage these interactions, the website includes primers on new techniques and interviews with the people developing them, technique validation and short video tutorials. We are also featuring case studies in which experts in microscopy and image analysis describe a problem that they have had, and how they went about solving it.

This is your site, so please use it! Read, post, comment, connect and feed back to us about what you like, what you don’t like, what’s missing, or what you’d like to see more of. We want to hear from you.

FocalPlane is supported by a distinguished Scientific Advisory Board, and will be run by Community Manager Dr Christos Kyprianou – you can read his welcome message here. Also check out Sharon Ahmad’s FocalPlane origin story. Like the Node, FocalPlane is a community site, so we encourage you to get involved – considering how fundamental microscopy is to developmental biology, we’re sure you’ll find it a useful resource.

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In this episode, supported by the Medical Research Council, we discover how researchers are letting the light shine in, literally, by bringing discoveries about the underlying genetic faults that cause eye diseases all the way through to game-changing clinical trials of gene therapy designed to save sight.

Our stay-at-home roving reporter Georgia Mills has been speaking with sight loss charity campaigner and fundraiser Ken Reid about his experiences of living with the genetic eye condition Retinitis Pigmentosa (RP).

She also chats to researchers Chloe Stanton and Roly Megaw from the MRC Human Genetics Unit in the Institute of Genetics and Molecular Medicine at the University of Edinburgh, who are researching the genes and mechanisms underpinning the disease, and Robin Ali at King’s College London who is running clinical trials of gene therapy for inherited eye disorders.

Genetics Unzipped is the podcast from The Genetics Society. Full transcript, links and references available online at GeneticsUnzipped.com

Subscribe from Apple podcasts, Spotify, or wherever you get your podcasts.

And head over to GeneticsUnzipped.com to catch up on our extensive back catalogue.

If you enjoy the show, please do rate and review on Apple podcasts and help to spread the word on social media. And you can always send feedback and suggestions for future episodes and guests to podcast@geneticsunzipped.com Follow us on Twitter – @geneticsunzip

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This post highlights the approach and findings of a new research article available in preprint on BioRxiv. This feature was written by members of the Solana lab, authors of that paper.

Single cell techniques are revolutionising biology, but at the moment they are largely limited to traditional model organisms and require access to specialised equipment in traditional laboratory settings. We are excited to introduce ACME, a novel technique which promises to democratise access to the world of single cell analysis, simplifying the extraction and processing of samples. We think this will be a ground-breaking innovation for cell biology, developmental biology and evo devo research.

ACME (when not written on an anvil in a Warner Bros cartoon), stands for ACetic acid-MEthanol dissociation. This principle stems from a 19th century approach for cell dissociation for microscopy. For instance, see its application in Hydra and in planarians. We show that, with modification, this same principle works for single cell transcriptomics, profiling the cellular atlases of two planarian species using SPLiT-seq.

What are the benefits of ACME dissociation?

An ongoing problem in single cell sequencing analysis is the time taken between sampling and fixing cells. During this time, cells are alive and under stress, which causes changes to their transcription and cellular identity. Nuclei based methods can get around this problem to an extent, but yield much lower RNA content and have their own technical requirements.

ACME cells are fixed from the time of dissociation and can be cryopreserved one or several times at several points throughout the process. This can be right after dissociation, for instance, in the field or when doing multi step protocols. In the lab, this can also be after FACS enrichment. This will allow exchange of samples between labs, preservation of fixed patient material, and freezing large sample sets for parallel analysis (avoiding some batch effects). RNA from ACME dissociated cells has high integrity, provided that RNAse-free conditions are met. Even after 5 cycles of freezing and thawing we recover RNAs with acceptable integrity – but one or two freezing steps should be enough for most applications.

ACME disassociation also preserves cell morphologies – this could be a game-changer by allowing a variety of integrated approaches to studying cellular evolution and homologies, which are not possible with most current cell fixation methods for scRNAseq.

In short, ACME greatly streamlines the preparation of single cells for scRNAseq, while allowing approaches to answer questions beyond the scope of current methods.

How is ACME dissociation performed?

ACME solution is made from common lab reagents, including (unsurprisingly) acetic acid and methanol. We show that this method efficaciously dissociates planarians in ~1h. Cells are then resuspended in PBS with BSA for molecular biology. We have successfully used ACME dissociation with animals including zebrafish, fruit flies, spiders, annelids, snails and sea anemones. Sometimes the harder outer layers of animals or embryos must be removed, but this is usually a simple process, and normally can be adapted from standard protocols.

We demonstrate a simple staining process for flow cytometry and FACS. This is based on a nuclear stain (DRAQ5) and a cytoplasmic stain (Concanavalin-A). This allows us to distinguish ACME dissociated single cells from aggregates and debris.

Test-driving ACME

To test that ACME dissociated cells can be used for combinatorial barcoding scRNAseq we performed SPLiT-seq in a species mixing experiment, including two cryopreservation steps. We managed to sequence 34K cells from the two species, with excellent species separation. Our models were the planarian Schmidtea mediterranea (which has been subject to scRNAseq before, allowing us to benchmark our results) and a second planarian species, Dugesia japonica (which hasn’t been sequenced, allowing us to prove that ACME copes with novel species just as well!).

In the planarian Schmidtea mediterranea we find markers of all previously described cell types, showing that ACME dissociation does not artificially deplete any known kind of cell. As a bonus, we find some new clusters, such as the nanos+ germ cell progenitors, which were known to exist, but had not been noted as distinct clusters in planarian scRNA seq data before. The early fixation provided by ACME potentially allowed these cells to be fixed quickly, while it is possible they lost their distinct expression profile in previous experiments.

We also describe for the first time the cell type atlas of a second planarian species, Dugesia japonica. This shows many initial similarities with Schmidtea mediterranea – the most abundant cell type groups in both species are epidermal, neural and muscular cells, present at comparable proportions. The depth of sampling, however, will allow us to compare even rarer cell type abundance and gene expression, allowing species vs species comparison at excellent resolution, even off a single cell sequencing run.

Future possibilities, and please try it out!

We plan to use ACME to explore cell type evolution in a variety of non-model organisms, However, it could also be of broad use in more traditional model organisms, particularly when fixing multiple samples for simultaneous analysis. While we use SPLiTseq, there is no conceptual reason why it would not also work for droplet-based techniques, and could be adapted to solve many problems in current single cell sequencing protocols. Further optimisation will of course continue to take place, and we will be happy to offer advice to anyone willing to try it in their own odd organism.

In short, ACME is a versatile and powerful cell dissociation method for single-cell transcriptomics, providing early fixation and easy storage of material. This will greatly enhance investigations of cell type diversity and dynamics in multiple different organisms – particularly in challenging conditions.

This work was lead by Helena García-Castro and Jordi Solana, with Nathan Kenny, Patricia Álvarez-Campos and Vincent Mason from the Solana Lab, Oxford Brookes, and collaborators from across the UK and Europe (Anna Schonauer, Vicky Sleight, Jakke Neiro, Aziz Aboobaker, Jon Permanyer, Marta Iglesias, Manuel Irimia and Arnau Sebé-Pedrós). Our thanks to the Node for letting us share it with you here.

This is the first paper from Jordi’s new lab at Oxford Brookes – more coming soon. And we will be soon recruiting a bioinformatician. Please contact Jordi Solana if this sounds like you!

References:

• Baguñà, J. and Romero, R., 1981. Quantitative analysis of cell types during growth, degrowth and regeneration in the planarians Dugesia mediterranea and Dugesia tigrina. Hydrobiologia, 84(1), pp.181-194. https://link.springer.com/article/10.1007/BF00026179
• David, C.N., 1973. A quantitative method for maceration of Hydra tissue. Wilhelm Roux Archiv für Entwicklungsmechanik der Organismen, 171(4), pp.259-268. https://link.springer.com/article/10.1007%2FBF00577724
• García-Castro, H., Kenny, N.J., Álvarez-Campos, P., Mason, V., Schönauer, A., Sleight, V.A., Neiro, J., Aboobaker, A., Permanyer, J., Iglesias, M. and Irimia, M., 2020. ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics. bioRxiv. https://www.biorxiv.org/content/10.1101/2020.05.26.117234v
• Plass, M., Solana, J., Wolf, F.A., Ayoub, S., Misios, A., Glažar, P., Obermayer, B., Theis, F.J., Kocks, C. and Rajewsky, N., 2018. Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics. Science, 360(6391). https://science.sciencemag.org/content/360/6391/eaaq1723
• Rosenberg, A.B., Roco, C.M., Muscat, R.A., Kuchina, A., Sample, P., Yao, Z., Graybuck, L.T., Peeler, D.J., Mukherjee, S., Chen, W. and Pun, S.H., 2018. Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding. Science, 360(6385), pp.176-182. https://science.sciencemag.org/content/360/6385/176
• Stuart, T. and Satija, R., 2019. Integrative single-cell analysis. Nature Reviews Genetics, 20(5), pp.257-272. https://doi.org/10.1038/s41576-019-0093-7

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