Review coordinated by Life Science Editors.

Reviewed by: Dr. Helen Pickersgill, Life Science Editors

Potential Conflicts of Interest: None

Main point of the paper: By combining multiple stains and antibodies with ultrastructural expansion (light) microscopy in Plasmodium falciparum during the course of mitosis within red blood cells (the asexual blood stage), when it causes the symptoms of malaria, the authors identified new structural features of cell division in this important human parasite.

Why this is interesting: Imaging the dramatic physical events that occur when cells divide tells us a lot about the biology of the process and is insightful to compare between different eukaryotes, but many organisms are too small to visualise by light microscopy, which is the most versatile imaging technique. So, they used an existing preparation technique to enlarge the parasites, a wide array of dyes and antibodies, and sampled at multiple timepoints so that more intracellular structures could be visualised and their behaviour and potential functions in cell division revealed.

Background: Expansion microscopy (ExM) has been around since 2015 (doi: 10.1126/science.1260088) and is a fairly simple and affordable technique. It involves physically magnifying a specimen by embedding it in a polyelectrolyte gel that swells up in water enabling super-resolution imaging. It has been previously applied to Plasmodium and other Apicomplexa, but not with so many different labels across different timepoints at this important life-stage.

Results: • They imaged 13 subcellular structures (including microtubules, microtubule organising centres, apicoplasts, Golgi and the ER) at multiple timepoints covering the entire asexual blood stage. • Among many results were the following: • They found a central role for the nuclear MTOC in coordinating mitosis and likely in establishing apical-basal polarity early during the asexual cycle. o the MTOC is tethered to the parasite plasma membrane (via cytoplasmic extensions) throughout mitosis. o the cytoplasmic extensions of the MTOC were closely associated (in numbers and positions) with several apical structures including the Golgi and the basal complex. o the MTOC is tethered to the mitochondria and apicoplast during fission and may also regulate their copy numbers. • They performed the first detailed characterization of the short-lived interpolar spindles, previously difficult to visualize, which consist of microtubules connecting duplicated MTOCs as they move to opposite sides of the cell. They found a large variation in their size, which may be how they enable the MTOCs to move without detaching from the plasma membrane. • They were able to study the biogenesis of rhoptries, which secrete proteins required for the parasite to invade host red blood cells, and discovered that: o biogenesis begins earlier than thought and that they associate with MTOCs during the remaining rounds of mitosis. o Rhoptry pairs are likely synthesized independently because over 95% are different sizes and densities.

Remaining thoughts: • Clearly written and easy to read paper with stunning images. • Comprehensive (including descriptions of when things didn’t work), but remains largely descriptive (of course). • Could be shortened/sharpened to make it more manageable to read without losing the main messages, which are somewhat lost in the text. • A top-level, specialised paper that opens the door for more targeted studies of organelle functions during mitosis and comparisons of these functions with other (higher) eukaryotes. • By identifying key players in mitosis during the asexual blood stage it may reveal candidate therapeutic targets for treating malaria.

Review coordinated by Life Science Editors. Reviewed by: Dr. Helen Pickersgill, Life Science Editors Potential Conflicts of Interest: None

Main point of the paper: The authors discover a new function for a nucleolar protein, a hydroxylase called MINA (a Myc target gene) involved in ribosome biogenesis and linked with lots of diseases (cancer, allergy), at the plasma membrane of epithelial cells. Here, it promotes epithelial tight junction integrity and barrier function, likely together with a guanylate kinase, MPP6.

Why this is interesting: This may be the first link specifically between ribosome biogenesis and adhesion, which occur in two different subcompartments of the cell, and it is mediated by one protein, MINA. The linking of these processes would enable the careful coordination that is needed between cell division and adhesion, and could thereby help cells transition between two states (i.e., from quiescence to proliferation). So, it’s the multifunctional property of MINA in two different parts of the cell that could help promote an important state transition, which may be why it’s linked to different diseases.

Background: MINA is primarily known as a nucleolar-localized protein that promotes ribosome biogenesis/function. It catalyzes histidine hydroxylation of the large ribosomal subunit protein RPL27A thereby promoting its activity. Consistent with this, MINA is upregulated in rapidly growing cells, including tumors. However, it is expressed also in normal human tissues, and has also been linked with seemingly ribosome-independent functions, specifically in suppressing cell migration, invasion, and EMT. This suggests it has other, unknown functions, which may or may not be related to each other.

Transitioning between distinct cell states such as differentiation and proliferation requires extensive coordination of diverse processes. For example, transitioning from quiescence to growth requires increasing metabolic activity and involves coupling processes like the cell cycle and adhesion to enable the cell to successfully divide.

Results: • MINA depletion in intestinal epithelial cells (Caco-2 cells stably expressing MINA-targeting shRNA) caused flattening and other phenotypes indicative of a role in tight junction integrity and barrier function. This pointed to an unexpected role for a nucleolar protein in epithelial membrane biology. • Pull-downs with a MINA mutant that was unable to localize to the nucleolus in three cell lines (Caco-2, U2OS, and MEFs) together with proteomic screens in Caco-2 cells of the WT and nucleolar-interacting mutant identified MPP6, a member of the MAGUK superfamily of cell adhesion and polarity proteins, as a candidate interacting partner of MINA. • In vitro interaction assays with endogenous and tagged proteins verified the MINA-MPP6 interaction in the nucleoplasm, and showed that it was promoted by RNApol1 inhibition (which increases the pool of extra-nucleolar MINA in subconfluent cells).<br /> • MINA knockdown redistributes HA-tagged-MPP6 from the plasma membrane to the nucleus, suggesting a functional consequence of this interaction. • shRNA knockdown of MPP6 phenocopied MINA depletion with respect to altered barrier function. i.e., both are implicated in tight junction integrity and epithelial barrier function.

Remaining questions/points: • It’s not yet demonstrated definitively that MINA works via MPP6 in tight junction integrity and epithelial barrier function. It would help to have more insight into this mechanism – it appears to not be related to MINA’s catalytic activity, and may ‘just’ involve MINA promoting MPP6 membrane localization, but whether this is direct or indirect (and if/how it’s regulated) is unclear. • Whether there is a regulatory link between ribosomal biogenesis and membrane biology via MINA is unclear – i.e., if/how MINA’s two functions are physiologically regulated to coordinate proliferation and adhesion (this may require going beyond cell lines). It could be that these two functions are normally independent (but if you experimentally interfere in vitro, then you see a dependency).

Review coordinated by Life Science Editors

Reviewed by: Dr. Helen Pickersgill, Life Science Editors

Potential Conflicts of Interest: None

Impact Point: Transcription factors may mediate the release of specific genes in extracellular vesicles that could be taken up by other cells and directly influence their behaviour.

Background: Extracellular vesicles (EVs) are nanoscale, membrane-bound vesicles containing proteins, nucleic acids and lipids that are released by most cell types. Originally thought of as a cell waste disposal mechanism, they have since been rebranded as a means of intercellular communication, both short and long range, (like a cellular postal service), and can, for example, modify gene expression and function in recipient cells via the transfer of specific RNAs (DOI: 10.1038/s41580-020-0251-y). One interesting function for EVs was the recent discovery that antigen presenting cells (APCs) could donate telomeres via EVs to recipient T cells and rescue them from senescence (DOI: 10.1038/s41556-022-00991-z).

Given EVs contain a wide variety of cargos derived from the secreting cells, which have been extensively profiled (e.g., DOI: 10.1016/j.cell.2020.07.009) they are particularly interesting as sources of biomarkers for diagnosing diseases such as cancer (e.g., DOI: 10.1038/s12276-019-0219-1). We might also be able to use them as stable delivery mechanisms for controlling cell behaviour or targeting therapeutics/diagnostics.

We know quite a bit about how RNAs and proteins are selected for secretion by EVs (mediated by the autophagy protein LC3: e.g., DOIs: 10.1080/15548627.2020.1756557; 10.1038/s41556-019-0450-y). But little is known about DNA. DNA presents a particular challenge as it is packed up into the nucleus. This is also particularly important to understand in the context of horizontal gene transfer, i.e., passing functional genes between cells.

Main question: How does a cell ‘select’ specific DNA cargo from the nucleus and enable it to be released by EVs?

The advance: They discover that a transcription factor (FOXM1) plays a central role in mediating DNA release in EVs.

Results: • FOXM1 and LC3 (autophagy protein) colocalize and interact in cultured cells (coIP endogenous and exogenous, EMSA, immunostaining and identify an FOXM1-binding domain mutant). • FOXM1, LC3 and DNA colocalise in the cytoplasm in cultured cells, which increases upon starvation-induced autophagy (immunofluorescence). • FOXM1 and LC3 are found in EVs released from cultured cells (Western blot). • 15,544 DNA identified in EVs released from cultured cells (evDNA sequencing), of which 25 overlapped with DNA loci binding FOXM1 (ChIP). • FOXM1-bound nuclear DNA is transported to the cytoplasm upon induction of autophagy and is released in EVs in cultured cells (knock-in tagged chromatin with CRISPR-cas9, IF and PCR). This is dependent on FOXM1 (knock-out in cultured cells).

Significance: Transcription factors display strong DNA binding specificity and so are ideal candidates for directing specific genes into EVs for potential transfer to recipient cells.

Remaining questions/points: Care needs to be taken with regard to purification of EVs. Are the FOXM1-DNAs in the EVs functional in recipient cells? Is the DNA being actively ‘selected’ for an intercellular signalling purpose or is this just random degradation? Is it all FOXM1 bound DNA that has the potential to be trafficked to EVs or just a subset? Do other transcription factors have the same function or is it specific to this protein/family? Does this mechanism occur in other contexts (e.g., in vivo, under disease conditions).

Review coordinated by Life Science Editors.

Reviewed by: Dr. Angela Andersen, Life Science Editors

Potential Conflicts of Interest: None

Background: The ability to reproduce decreases with age in many animals - including humans and worms. Oocytes age earlier than other tissues, and their decline in quality contributes to reduced reproduction. Diminished mitochondria number/activity in human oocytes correlates with age-related decline.

Question: Does mitochondrial dysfunction cause (or just correlate with) reduced egg quality?

Summary: The authors compared the proteomes of mitochondria isolated from young worms, old worms and daf-2 mutant worms (c. elegans) (insulin/igf-1 receptor mutant) that have longer lifespans & longer reproductive lifespan. * Mitochondria from young & mutant worms had high levels of BCAT-1 (branched chain aminotransferase). * RNAi of bcat-1 reduced the longevity, reproductive longevity & egg quality of daf2 mutants, and increased mitochondrial activity/mtROS. * Similar effects of bcat-1 kd in wt worms, but interestingly the effects on reproductive longevity were more severe in wt than daf2 mutants (from a quick look), but there was no effect on lifespan in wt animals. * Overexpressing bcat-1 in wt extended reproduction & egg quality but not lifespan. * Treating animals with vitamin B1 (a cofactor downstream of BCAT1 in BCAA metabolism) delayed reproductive aging, slightly extended lifespan, improved oocyte quality, reduced mtROS in aged worms..

Advance: BCAT-1 levels/BCAA metabolism correlate with mitochondrial quality & reproductive longevity. Vit B1, which promotes BCAA metabolism, can extended reproductive longevity.

Significance: More/strong evidence that dysfunctional mitochondria cause a decline oocyte quality, reduce reproductive longevity. If vitamin B1 supplements are a safe way to delay age-related decline of eggs in female mammals (humans) that would be amazing. .

Ang asks:

• is this effectively dietary restriction of BCAA? Would that be a better (albeit perhaps more difficult to sustain) approach?

• How does this relate to some recent papers pointing out that mitochondria in eggs are special (e.g. Cheng et al. Mammalian oocytes store mRNAs in a mitochondria-associated membraneless compartment, Science 2022; Rodriguez-Nuevo et al., Oocytes maintain ROS-free mitochondrial metabolism by suppressing complex I, Nature 2022) and a role for BCAA in longevity (e.g. Richardson et al., Lifelong restriction of dietary branched-chain amino acids has sex-specific benefits for frailty and life span in mice, Nature Aging 2021).

• How does low BCAA metabolism lead to mitochondrial dysfunction/oocyte aging? Is it related to accumulation of amino acids in the cytosol and toxicity to mitochondria? (e.g. Hughes et al., Cysteine toxicity drives age-related mitochondrial decline by altering iron homeostasis, Cell 2020).

• Overall these data support the idea that oocytes are particularly vulnerable to conditions that drive aging, and conserved aging mechanisms in the soma and germline as well as across species.

Review coordinated by Life Science Editors.

Reviewed by: Dr. Angela Andersen, Life Science Editors

Potential Conflicts of Interest: Dr. Mill has worked with Life Science Editors on other manuscripts.

Background: Retinitis pigmentosa (RP) is a group of rare eye diseases that cause vision loss. Symptoms usually start in childhood, and most people eventually lose most of their sight. There is no cure for RP. Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause RP and compromise the renewal of light-sensitive “disc” membranes (specialized cilia) at the outer segment of photoreceptors, resulting in the loss of these cells over time. Evidence suggests that disc formation is similar to the release of ectosomes (small extracellular vesicles) and that both rely on the actin cytoskeleton. Knockdown of RPGR in retinal pigmented epithelium cells showed stronger actin filaments and reduced cilia suggesting that it may regulate nascent photoreceptor disc formation by regulating actin-mediated membrane extension in the retina (Gakovic et al., Human Molecular Genetics, 2011). In addition, RPGR patient iPSC-retinal models displayed phenotypes consistent with abnormal actin regulation (Megaw et al., Nature Communications, 2017; Karam et al., J Personalized Medicine, 2022).

Question: What function of RPGR is compromised in photoreceptors to cause RP?

Advance: The authors generated novel Rpgr mutant mice harboring human disease-causing mutations that recapitulate human disease phenotypes: aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. RPGR is located at the site of disc formation – to test if it plays a role in disc genesis, they engineered a novel reporter mouse to track outer segment turnover. Rhodopsin was tagged with the self-labelling peptide SNAP- Rhodopsin is the major protein component of outer segment discs, and so incubating RhodSNAP retinal slice cultures with SNAP fluorophores results in outer segment labelling. Perturbation of RPGR resulted in a slowed rate of disc formation, leading to shortened outer segments and increased vesicle shedding. To me, the breakthrough is in the last figure: the actin depolymerizing drug Cytochalasin D in PBS was injected intravitreally, and fixed retinas were analyzed 6 hours later by electron microscopy. Cytochalasin D treatment significantly reduced the number of shed vesicles from the base of the outer segment in Rpgr-mutant mice (they now look like wild-type).

Significance: Nails down the disease-relevant function of RPGR and a molecular mechanism of RP in photoreceptor cells, in vivo, in mice. Pharmacological rescue not only demonstrates the importance of the mechanism to disease but also sheds light on a potential therapeutic avenue for RP.