A distinct phase of cyclin B (Cdc13) nuclear export at mitotic entry in Schizosaccharomyces pombe

Abstract

In eukaryotes, cell division requires coordination between the nucleus and cytoplasm. Entry into cell division is driven by cyclin-dependent kinases (CDKs), which need a cyclin binding partner for their activity. In Schizosaccharomyces pombe (fission yeast), the B-type cyclin Cdc13 is essential and sufficient for cell cycle progression and is strongly enriched in the nucleus. Here, we show that a fraction of Cdc13 is exported from the nucleus to the cytoplasm just prior to mitosis. This export could be critical to propagate CDK activity throughout the cell. Mutating three Cdc13 nuclear localization signals (NLSs) led to precocious enrichment of Cdc13 in the cytoplasm but did not accelerate mitotic entry, indicating that the export is not sufficient to trigger entry into mitosis. The export coincides with spindle pole body integration into the nuclear envelope and may be required to coordinate nuclear and cytoplasmic signalling required for this integration. The onset and stop of Cdc13 nuclear export are remarkably abrupt, underscoring that S. pombe mitotic entry consists of several switch-like transitions over the course of minutes. Our findings add another instance to the various cyclin nuclear transport events known to occur at critical cell cycle transitions throughout eukaryotes.

Headnote

In eukaryotes, cell division requires coordination between the nucleus and cytoplasm. Entry into cell division is driven by cyclin-dependent kinases (CDKs), which need a cyclin binding partner for their activity. In Schizosaccharomyces pombe (fission yeast), the B-type cyclin Cdc13 is essential and sufficient for cell cycle progression and is strongly enriched in the nucleus. Here, we show that a fraction of Cdc13 is exported from the nucleus to the cytoplasm just prior to mitosis. This export could be critical to propagate CDK activity throughout the cell. Mutating three Cdc13 nuclear localization signals (NLSs) led to precocious enrichment of Cdc13 in the cytoplasm but did not accelerate mitotic entry, indicating that the export is not sufficient to trigger entry into mitosis. The export coincides with spindle pole body integration into the nuclear envelope and may be required to coordinate nuclear and cytoplasmic signalling required for this integration. The onset and stop of Cdc13 nuclear export are remarkably abrupt, underscoring that S. pombe mitotic entry consists of several switch-like transitions over the course of minutes. Our findings add another instance to the various cyclin nuclear transport events known to occur at critical cell cycle transitions throughout eukaryotes.

Keywords: cell cycle, cyclin B, mitotic entry, nucleocytoplasmic transport, Schizosaccharomyces pombe

1. Background

The eukaryotic cell cycle is characterized by distinct phases (G1, S, G2 and M) with rapid transitions from G1 to S and G2 to M. The key drivers of the cell cycle are cyclin-dependent kinases (CDKs), which require binding to a cyclin protein for their activity [1-3]. In Schizosaccharomyces pombe (fission yeast), the cyclin-dependent kinase CDK1 (S.p. Cdc2) is essential for cell cycle progression and can pair with several cyclins [4]. The B-type cyclin Cdc13 is the only one of these cyclins that is essential for viability, and Cdc13 is sufficient to execute the cell cycle in the absence of the other Cdc2-binding cyclins [5,6]. Cdc13 accumulates during the S and G2 phases of the cell cycle and is ubiquitinated by the anaphase-promoting complex (APC/C) late in mitosis and G1, which targets it for proteasomal degradation. In all cell cycle phases where Cdc13 is present, it is strongly enriched in the nucleus [7-12].

The localization of cell cycle cyclins (and their CDK binding partners) is often dynamic [13]. S.p. Cdc13-in addition to its nuclear localization - binds to spindle pole bodies (SPBs) starting in late G2 phase and to the mitotic spindle during mitosis [12,1419]. Cdc13 can also become enriched in the nucleolus at times [20-23]. In mammalian cells, cyclin A2 is mostly nuclear but becomes enriched in the cytoplasm after S phase and at centrosomes (equivalent to yeast spindle pole bodies) in early mitosis [24-31]. Mammalian cyclin В1 is mostly cytoplasmic but also localizes to centrosomes and is rapidly imported into the nucleus at entry into mitosis |2 | [24,25,32-34]. Cyclin B1's nuclear import propagates CDK1 activity into the nucleus [34,35] and creates a spatial positive feedback loop, supporting a rapid and irreversible transition from G2 phase to mitosis [36,37]. In mitosis, cyclin B1 also localizes to spindle microtubules and kinetochores [24,32,36,38-41].

Here, we report that a fraction of 5. pombe Cdc13 becomes exported from the nucleus just prior to mitosis, which has recently also been described by Kapadia & Nurse [42]. We find that Cdc13 export starts concomitant with Polo-like kinase (Plo1) enrichment at SPBs and stops when spindle pole bodies separate and the mitotic spindle forms. Along with other observations [42-48], our findings indicate that 5. pombe mitotic entry is a fast sequence of at least three distinct switch-like events, probably coupled to the integration of the SPBs into the nuclear envelope.

2. Results

2.1. Cdc13 and Cdc2 export from the nucleus prior to degradation of Cdc13 in mitosis

When performing live-cell imaging of Cdc13-sfGFP, we observed a drop in nuclear concentration (figure 1A, black arrows) that occurred just prior to mitosis and distinctly prior to the degradation of Cdc13 in late mitosis (figure 1A, grey arrows). This drop is too subtle to be observed in population data when cells are arranged by size (electronic supplementary material, figure S1) [18] but has also been observed by others using live-cell imaging, either with the same or another Cdc13 tag [42,48]. The drop in nuclear concentration was initially interpreted as reflecting movement of Cdc13 to the SPB [48]. While SPB localization is visible in this period (figure 1B, arrowheads), we also detect a more general increase in cytoplasmic Cdc13 concentration in the same period, whereas the cellular concentration of Cdc13 stayed comparatively constant (figure 1A-C). Highly similar measurements were made by Kapadia & Nurse [42]. When we plotted the total amounts of Cdc13 in the cell, nucleus and cytoplasm, rather than the concentration, the cytoplasmic amount increased faster than the total amount and concomitant with a decrease in the nuclear amount (figure 1D). This indicates that a fraction of Cdc13 is exported from the nucleus at this time, prior to Cdc13 degradation in late mitosis (figure 1E). (Note that what we call "export" could either be export or less efficient import of Cdc13 that cycles between nucleus and cytoplasm.)

Cdc13 binds Cdc2 and is required for the nuclear localization of Сас2 [7,8,12]. We found that Cdc2 nuclear concentration also started to drop prior to mitosis, accompanied by an increase in cytoplasmic concentration (figure 2A, black arrowheads; electronic supplementary material, figure S2). An additional, further decrease in nuclear and increase in cytoplasmic Cdc2 concentration is observed later, when Cdc13 is degraded and no longer available as a binding partner (figure 2A, grey arrowheads; electronic supplementary material, figure 52). This suggests that Сас13 and Cdc2 are exported from the nucleus as a complex, and therefore that CDK1 activity may spread to the cytoplasm during this time. As a control, another nuclear-enriched protein (Mad3, a spindle assembly checkpoint protein) did not show a drop in nuclear concentration during that same period (figure 2A; electronic supplementary material, figure 52), indicating that the export is actively regulated rather than representing unspecific leakiness of the nuclear envelope. Leakiness may have occurred because the S. pombe SPB integrates into the nuclear envelope during this time, which requires nuclear envelope fenestration [49,50].

Even though Cdc13 and Cdc2 may export as a complex, the export of Cdc13 does not require Cdc2, since the N-terminal unstructured region of Cdc13, Cdc13-(1-177), which is not expected to interact with Cdc2 [38,51,52], shows the same brief period of nuclear export (figure 2B). (Note that Cdc13-(1-177) is overexpressed in this experiment, making the increase in cytoplasmic concentration more obvious.)

2.2. Cdc13 export takes place prior to spindle pole body separation and is concomitant with Plo1 enrichment at spindle pole bodies

To better assess the timing of Cdc13 export, we co-labelled the centromere of chromosome 1 with tdTomato, took images every 15 s and aligned the measurements to sister chromatid separation in anaphase (figure 3, electronic supplementary material, figure 53). Nuclear export of Cdc13 began about 15 min prior to anaphase and, remarkably, ceased about 7 min prior to anaphase, which corresponds to the time when spindle poles separate. Another APC/C substrate, securin (S.p. Cut2), showed a different pattern: while Cdc13 was exporting, Cut? nuclear concentration stayed constant, but it imported into the nucleus starting at SPB separation (figure 3). This confirms that Cdc13 nuclear export is not a consequence of nuclear leakiness and indicates two different phases of nuclear transport regulation at entry into mitosis: before and after spindle pole separation.

At this higher temporal resolution, it became apparent that the onset and stop of nuclear export are rapid events, observable by the abrupt slope change both in the averaged data (figure 3) as well as in single cells (electronic supplementary material, figure 54). The transition to decreasing nuclear Cdc13 concentration happens within about a minute, and the transition from export to its cessation is typically even more abrupt (electronic supplementary material, figure 54). This suggests that the onset and stop of nuclear export are switch-like transitions and require a type of molecular regulation that can implement such rapid change.

To position the onset of Cdc13 nuclear export relative to other events, we looked at Plo1 localization. Plo1 (Polo or PIk1 in other organisms) is a key kinase in the regulation of mitotic entry in both mammalian cells and S. pombe [53-57]. In 5. pombe, Plo1 becomes enriched at SPBs in late G2, just prior to mitosis, and SPBs serve as a signalling hub for mitotic entry regulation involving Plol and Cdc2 [12,17,19,57-61]. We imaged Plo1-mCherry together with Cdc13-sfGFP. In these experiments, Cdc13-sfGFP was expressed from the endogenous locus either under the endogenous cdc13 promoter or overexpressed under the adh1 promoter (figure 4A). These experiments confirmed that Cdc13 nuclear export takes place prior to SPB separation and showed that its start is concomitant with the enrichment of Plo1 at SPBs and stops with SPB separation (figure 4A,B). Fission yeast SPBs are located outside the nuclear envelope during interphase and need to integrate into the nuclear envelope for mitotic spindle formation [49,50,62,63]. The coincidence in timing suggests that Cdc13 export is important for SPB integration.

2.3. Movement of Cdc13 from nucleus to cytoplasm is not sufficient for mitotic entry

To address whether the export of Cdc13 from the nucleus influences the timing of entry into mitosis, we sought to precociously enrich Cdc13 in the cytoplasm. The N-terminal unstructured region of Cdc13 contains three candidate nuclear localization signals (NLSs) (figure 5A, electronic supplementary material, figure S5) [8]. We mutated these and expressed the mutant constructs under the endogenous regulatory sequences from an exogenous locus, leaving the endogenous cdc13 intact. While mutation of individual NLSs did not prominently affect Cdc13 nucleocytoplasmic distribution (figure 5B,C, and not shown), combining mutations in all three NLSs led to a reduction in nuclear enrichment (figure 5B,C, NLS1-2-3 mutant). We initially left two lysine residues that are part of the predicted bi-partite NLS] intact because they are directly adjacent to the destruction box (D-box), and we assumed they may be important targets for ubiquitination and therefore essential for the rapid proteasomemediated degradation of Сас13 in late mitosis. However, mutation of these residues still allowed for degradation of Сас13 at the end of mitosis while further impairing nuclear enrichment (figures 5B,C and 6A, electronic supplementary material, figure S6A,B, NLS-KK mutant). In live-cell imaging, a prominent cytoplasmic pool of Cdc13-NLS-KK was observed throughout the cell cycle (figure 6A, electronic supplementary material, figure S6B). Despite the precocious and strong enrichment of Cdc13-NLS-KK in the cytoplasm, the length of the cell cycle was not shortened in this strain (figure 6B), indicating that Cdc13 nuclear export is not sufficient to trigger mitotic entry. This situation is similar to that of mammalian cyclin B1, which rapidly imports into the nucleus at entry into mitosis, but in cell lines, a constitutive or precocious nuclear localization of cyclin B1 does not accelerate entry into mitosis [64-67].

Consistent with the unaltered cell cycle length, we found that strong enrichment of Plo1 at SPBs remained restricted to approximately 8 min prior to SPB separation in cells expressing Cdc13-NLS-KK (figure 7, electronic supplementary material, figure 57). Plo1 enrichment still coincided with Cdc13 nuclear export. Nuclear export was detectable as a drop in nuclear signal in the Cdc13-NLS-KK mutant despite its lowered nucleocytoplasmic ratio (figure 7, electronic supplementary material, figure S7). This suggests that the change in nucleocytoplasmic Сас13 distribution prior to mitosis is not a consequence of blocking NLS function -at least not of those NLSs that are mutated in Cdc13-NLS-KK. What mediates the remaining nuclear enrichment of Cdc13-NLS-KK is unknown. Cdc13 does not contain other classical NLSs. Nuclear accumulation of Cdc13 therefore may partially rely on non-canonical mechanisms such as direct binding to importin-B, as is the case for other cyclins [27,68-70].

Taken together, the unaltered cell cycle length and similar kinetics of Plo1 accumulation at SPBs in Cdc13-NLS-KK-expressing cells suggest that increasing CDK1 activity in the cytoplasm is insufficient to trigger mitotic entry.

While the cell cycle length of Cdc13-NLS-KK mutant-expressing cells was unchanged, the size of these cells increased (figure 6C; electronic supplementary material, figure S6C). Size homeostasis (longer cell cycles in cells that are born shorter [71]) was maintained (figure 6D; electronic supplementary material, figure S6D,E), indicating that the "set point' for size had changed without altering the regulation of size control. We attribute this to the altered distribution of Cdc13 (and therefore probably Cde?) between the nucleus and cytoplasm. With less nuclear CDK1 activity, cells may reach the threshold for mitotic entry only at a larger size. The alteration of cell size is consistent with observations that perturbing the nucleocytoplasmic ratio of the Cdc2 regulators Weel or Cdc25 alters cell size [45,72,73].

3. Discussion

The nucleus of eukaryotic cells provides additional opportunities for the regulation of cellular activities but also brings about the need to coordinate nuclear and cytoplasmic events. Changes in the nucleocytoplasmic distribution of cyclins or other CDK regulators are major themes in cell cycle regulation. Here, we show that a pool of 5. pombe Cdc13 translocates from the nucleus to the cytoplasm at about the time when Plo1 starts to become strongly enriched at SPBs. We suggest that this is part of a sequence of events leading to the insertion of the SPBs into the nuclear envelope and, ultimately, mitotic spindle formation (figure 8). The export could serve to spread CDK1 activity into the cytoplasm, as also proposed by Kapadia & Nurse [42], who have shown, using biosensors for CDK1 activity, that CDK1 becomes active in the nucleus prior to Cdc13 export and prior to detectable CDK1 activity in the cytoplasm. The Cdc13 translocation seems functionally analogous to the movement of vertebrate cyclin B1 at entry into mitosis [34], except that the directionality is reversed (vertebrate cyclin B1 moving from the cytoplasm to the nucleus, and 5. pombe Cdc13 moving in the other direction).

3.1. The switch-like onset and stop of Cdc13 nuclear export could be implemented by phosphoregulation

The mechanism by which the change in nucleocytoplasmic distribution of Cdc13 occurs is still unknown. One possibility is post-translational regulation, such as phosphorylation, which would be dynamic enough to change the nucleocytoplasmic distribution within minutes. The onset and stop of Cdc13 export are strikingly switch-like (figure 3; electronic supplementary material, figure S4), which suggests ultrasensitivity in the underlying regulation and could, for example, be implemented by multi-site phosphorylation or enzyme saturation [74,75]. Multiple phosphorylation sites have been identified in the N-terminal, unstructured region of Cdc13 that also contains the NLSs [22,7681] (electronic supplementary material, figure S5A), but the role of most of these sites has not yet been tested. Mutation of three phosphorylated Cdc13 residues downstream of the NLSs (S177, 5180 and 5183) did not interfere with Cdc13 localization or function [22]. It is also possible that import or export regulators are post-translationally modified. For vertebrate cyclin B1's rapid import into the nucleus at entry into mitosis, phosphorylation of both cyclin B1 and transport effectors has been implicated [33,34,64,82-84]. In 5. pombe, the phosphorylation of several nucleoporins increases around M-phase, which could alter nuclear transport [79,85,86]. Furthermore, reducing the dosage of several nucleoporins has been shown to increase cell size in S. pombe, similar to cdc13, cdc2 and cdc25 mutants [87]. Whether this is related to altered Cdc13 nuclear export remains to be tested.

3.2. Cdc13 export may propagate CDK1 activity to the cytoplasm to coordinate stepwise SPB integration

SPBs and their vertebrate counterparts, centrosomes, are considered hubs of mitotic entry regulation [53,56,88]. In S. pombe, the SPBs are located adjacent to, but outside, the nuclear envelope during interphase and only become integrated into the nuclear envelope at the onset of mitosis [50,62,89]. Their proper integration is required for the formation of the mitotic spindle [43,63,90,91]. Plo1, localized to SPBs, is needed for the integration of the SPBs into the nuclear envelope [47,60,92,93], and Cdc2, in turn, is required for Plo1 localization and activity at SPBs [59,61,94,95]. We consider it plausible that Cdc2/Cdc13 export from the nucleus to the cytoplasm is required to initiate SPB integration into the nuclear envelope by co-positioning Plol and Cdc13/Cdc2 at the cytoplasmic SPBs (figure 8). Contact between centromeres and the nuclear envelope beneath the SPBs is also required for SPB insertion, and this signalling has also been suggested to involve Р101 and possibly Cdc2 [46,47]. Hence, both nuclear and cytoplasmic Cdc2 activity could be important, and Cdc13 nuclear export may be required to provide them both. Interestingly, mutation of the hydrophobic patch of Cdc13 has been shown to prevent not only Cdc13 enrichment in the SPB region [17,19] but also Cdc13 nuclear export [42], which suggests positive feedback between localized CDK1 activity at the SPB region and Cdc13 export. The binding partner for the hydrophobic patch has not been identified, and it is unclear whether this reflects a defect in a nuclear or cytoplasmic interaction.

The start of Cdc13 export coincides with Plo1 enrichment at the SPB region and its stop with SPB separation (figure 4). Live-cell imaging of mitotic entry regulators by Masuda et al. [45] has uncovered one additional switch-like step between the enrichment of Plo1 at SPBs and SPB separation: the CDK1-inhibiting kinase Weel and the kinesin Cut7 become enriched at SPBs about 3-4 min prior to SPB separation [45], clearly after the start of Plo1 enrichment at about 6-8 min prior to SPB separation (figure 8). We suggest that the first wave of binding (Plo1 and Cdc13) corresponds to preparing the SPB for insertion, and the second wave (Weel and Cut?) corresponds to insertion into the nuclear envelope, which makes the SPB accessible to nuclear proteins such as Weel. At SPB separation, Weel is removed from SPBs, and the microtubule-anchoring protein Msd1 binds [45]. This final step coincides with the cessation of Cdc13 export. Thus, 5. pombe mitotic entry consists of at least three switch-like transitions, tightly linked to SPB events and bracketed by Cdc13 nuclear export. We propose that the export is required to provide both nuclear and cytoplasmic activities for SPB integration.

4. Material and methods

4.1. Strains and growth

Strains with zfs1+:natR:P.adh31-tetR-tdTomato, dh1L:ura4+:tetO, cdc13-S177S-sfGFPcp, Padh1-cdc13-S177S-sfGFPcp, cdc2-GFP, mad3-ymEGFP, cut2-GFP and plo1-mCherry have been described previously [96-99]. Strains expressing- from an exogenous locus - only the N-terminus of Cdc13 (cdc13(1-177)-sfGFPcp) or Cdc13 NLS mutants were generated by cloning the respective fragments or mutant versions into a pDUAL vector [100] and integrating the vector at the leu1 locus. The proper sequence of the vectors was confirmed by Sanger or Nanopore whole-plasmid sequencing. Some strains have a Y66L mutation introduced into GFP, which makes GFP non-fluorescent. This allows us to visualize other proteins with GFP while maintaining the tag for comparability with other strains. In circularly permuted sfGFPcp, Y138 (LTYGV) is the residue corresponding to Y66 (FTYGV) in canonical GFP.

4.2. Growth conditions

Cells were grown in Edinburgh minimal medium (EMM, MP Biomedicals, 411003) at 30°C to a concentration of 8 x 10° - 1.5 x 107 cells mI". Leucine (0.2 mg ml") or adenine (0.15 mg ml") was added when required. Preconditioned medium (made by filtering EMM cultures) was added when cultures were diluted to low densities.

4.3. Time-lapse imaging

Time-lapse imaging was conducted on a DeltaVision Elite microscope equipped with an Olympus 60x/1.42 Plan-APO oil objective, LED illumination and a PCO edge sCMOS camera. Cells were kept at 30%C for the duration of imaging (EMBL environmental chamber). Cells were either mounted in u-Slide 8 well glass bottom chambers (Ibidi, 80827) coated with lectin (50 ug ml"; Sigma-Aldrich, 1.1395) or loaded into microfluidics chambers (Millipore-Sigma, Y04C or Y04T) that had been washed with EMM and prewarmed to 30°C. Media flow for the microfluidics chambers was controlled using a CellAsic ONIX2 microfluidics system. EMM supplemented with 50% preconditioned medium was perfused at 2 psi. After being loaded into the viewing chamber of the microfluidics plate, cells were left to acclimate for 3 h before starting imaging. When imaging in u-Slide chambers, cells were kept on the 30°C microscope stage for 15 min before starting imaging. Brightfield images were taken at the bottom or central slice of each field of view and fluorescence images were acquired using "optical axis integration" (sum projection) over a 3.6 um Z-distance. Fluorescence images in experiments shown in figures 2, 4, 6 and 7 were additionally deconvolved using SoftWoRx (GE Healthcare) software with three cycles of the ratio method (conservative).

4.4. Imaging of asynchronous cultures

Asynchronous cultures were either imaged at 30%C on a DeltaVision Elite microscope (see above) or at room temperature (approx. 22°C) on a Zeiss Axiolmager M1 equipped with Xcite Fire LED illumination (Excelitas), a Zeiss Plan-APO 63x/1.4 oil objective and an ORCA-Flash4.0LT sCMOS camera (Hamamatsu). Cells were pelleted at 3300 rcf for 1 min, mounted from the pellet onto slides, covered with #1.5 glass coverslips and immediately imaged. Z-stacks were collected and the slice corresponding to the midplane of the cell was used for the quantification of nucleocytoplasmic ratios.

4.5. Image analysis

For the experiments in figures 1, 2, 5 and 6, and electronic supplementary material, figures S2 and S6, brightfield images were used to segment individual cells using the YeaZ neural network with custom weights [101]. Masks were manually edited using the YeaZ graphical user interface as needed. Cells partially out of frame were eliminated from the analysis. A custom Fiji [102] script was used to load corrected YeaZ cell masks as regions of interest (ROIs). Nuclei were segmented in Fiji using tdTomato-NLS fluorescence and Otsu thresholding. A Gaussian blur was applied to smooth the nuclear edges. After manual checking and correction, nuclear ROIs were assigned to cellular ROIs based on position; and size and fluorescence intensity were quantified. Cytoplasmic area and integrated intensity were calculated by subtracting nuclear area and integrated intensity from the respective whole cell measurements. To determine nucleocytoplasmic ratios, the fluorescence intensity outside cells, measured as the mean from several manually drawn ROIs, was subtracted as background. (Note that this does not take autofluorescence into account, which is difficult to quantify. Both nuclear and cytoplasmic concentrations are, therefore, slightly overestimated.) To calculate nucleocytoplasmic ratios, nuclear GFP concentration (integrated signal by area) was divided by the respective cytoplasmic concentration and normalized within each experiment to the mean nucleocytoplasmic ratio of cells expressing wild-type Cdc13-sfGFP.

The experiment in figure 4 lacked a brightfield image for cell segmentation and a fluorescent marker for nuclear segmentation. Cells were, therefore, segmented using trainable Weka segmentation [103] based on the diffuse Plo1-mCherry signal. Cytoplasmic concentrations were measured by manually placing two ROIs into the cytoplasm, one on each side of the nucleus. Plo1 enrichment at the SPB region and Cdc13 nuclear intensity were estimated by calculating the mean of the 40 brightest pixels in the cell. For the experiment in figure 7 and electronic supplementary material, figure S7, cells were segmented manually. Plo1 enrichment at the SPB region and nuclear Cdc13-sfGFP were estimated by calculating the mean of the 40 brightest pixels in the cell. Raw data curves were smoothed using the loess function in R (span 0.3).

The Pomegranate image analysis pipeline [104] was used for the three-dimensional quantification of Cdc13-sfGFP shown in electronic supplementary material, figure 51.

Ethics. This work did not require ethical approval from a human subject or animal welfare committee.

Data accessibility. Supplementary material is available online [105].

Declaration of Al use. We have not used Al-assisted technologies in creating this article.

Authors" contributions. S.G.C.: conceptualization, formal analysis, investigation, writing-review and editing; J.M.R.: conceptualization, formal analysis, investigation; D.V.: formal analysis, investigation; W.W.: formal analysis, investigation; V.G.: software; S.J.R.: funding acquisition, software, supervision; S.H.: conceptualization, formal analysis, funding acquisition, investigation, supervision, writing - original draft.

All authors gave final approval for publication and agreed to be held accountable for the work performed therein.

Conflict of interest declaration. We declare we have no competing interests.

Funding. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers R35GM119723 and R35GM149565 (S.H.); V.G. was supported by SNSF grants CRSK-3_190526, 310030_204938 and CRSK-3_221036 awarded to S.J.R.

Acknowledgements. We are grateful to Liv Erickson for experimental help, Yoshinori Watanabe for strains and Douglas Weidemann for comments.

References

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