Fly clock, my clock, and lamin B receptor.

In the fruit fly Drosophila melanogaster, circadian rhythm was disrupted when the inner nuclear membrane protein lamin B receptor (LBR) was depleted from its clock neurons (Proc. Natl. Acad. Sci. USA 118, e2019756118. 2021; https://doi.org/10. 1073/pnas.2019756118 and Research 6, 0139, 2023; https://doi.org/10.34133/research.0139). Ordinarily, the clock proteinPERIOD (PER) forms foci close to the inner nuclear membrane in the circadian clock's repression phase. The size, number, and location of foci near the nuclear membrane oscillate with a 24-h rhythm. When LBR was absent the foci did not form. The PER foci bring per and other clock genes close to the nuclear envelope, where their transcription is silenced. Then, in the circadian clock's activation phase, the PER protein gradually gets degraded and the foci disappear. The clock genes, including per, relocate to the nucleus interior where they resume transcription. Rhythmic re-positioning of clock genes between nucleus periphery and interior, correlates with their repression and activation in the circadian cycle. Absence of LBR disrupted this rhythm. Phosphorylation of PER promoted the formation of foci whereas dephosphorylation by protein phosphatase 2A causedthem to disappear. LBR promoted focus formation by destabilizing the catalytic subunit of protein phosphatase 2A. The lbr gene is no stranger to this journal. The first hint that vertebrate LBR is also a sterol biosynthesis enzyme, specifically, a sterol C14 reductase, was reported here (J. Genet. 73, 33-41, 1994; https://www.ias.ac.in/article/fulltext/jgen/073/01/0033-0041). Mutations in the human Lbr gene cause a range of phenotypes--from the relatively benign Pelger-Huet anomaly to the perinatally lethal Greenberg skeletal dysplasia.Drosophila, like all insects, is a sterol auxotroph. The fly orthologue of vertebrate lbr genes encodes a protein (dLBR) that shares several properties with vertebrate LBR proteins, with one notable exception. While human LBR complemented theyeast Saccharomyces cerevisiae erg24 mutant which lacks sterol C14 reductase activity, dLBR did not (J. Cell. Sci. 117, 2015-28, 2004; https://doi.org/10.1242/jcs.01052). Despite not possessing sterol reductase activity, dLBR retains significant sequence homology with vertebrate LBRs which have this activity. An undergraduate summer trainee in my laboratory obtained early (unpublished) evidence that dLBR lost sterol reductase activity during evolution. She transferred adult drosophila flies to vials containing a medium made of agar, dextrose, and dried and powdered mycelium of the filamentous fungus Neurospora crassa. On medium made with wild-type mycelium, theflies mated, laid eggs, hatched larvae, and developed pupae which eclosed progeny adult flies. The life cycle was no different than on 'regular' fly food composed of agar, dextrose and yeast extract. However, on a medium made with mycelium from a sterol C14 reductase null mutant, the flies laid eggs which hatched and released larvae, but the larvae failed to pupate, and no adult progeny flies emerged. This was because the fly lacks a sterol C14 reductase. The wild-type sterol, ergosterol, is a precursor of the steroid hormone ecdysone needed for molting and metamorphosis. Can expression of vertebrate LBR in dLBR-depleted fly clock neurons restore circadian rhythm? Can expression of vertebrate LBR enable flies to complete their life cycle on mutant Neurospora medium? Does LBR regulate the vertebrate clock in a like manner? If yes, then is the sterol reductase activity dispensable in this role? These are some questions that came to my mind on a recent morning walk. The walk itself was a much-cherished outcome of my circadian clock.

An evolutionarily conserved mechanism underlies interspecies cell-cell signalling in fungi.

When a conidium (vegetative spore) or ascospore (sexually produced spore) of the filamentous fungus Neurospora crassa germinates, it produces a long narrow filamentous multinucleate cell called a hypha. Hyphae grow by elongation, they can branch, and the tips of branches can also rejoin by fusion. Growth, branching, and fusion create an interconnected web, called a mycelium, within which cytoplasmic continuity is maintained. Some researchers have focused their studies on hyphal elongation and branching, others on formation of conidia and ascospores, and still others, prominently Andre Fleißner, Nick Read, Louise Glass, and colleagues, on tip fusion. Each of these fundamental processes contributes to the development of species-characteristic mycelial morphology. Using the fluorescently tagged Neurospora proteins MAK-2 and SO, they made the startling discovery that when tips of freshly germinated and genetically identical conidia (germlings) came within 15 µm of each other, each tip took turns to send and receive a molecular signal in an oscillatory back-and-forth manner.

Dictyostelium's pisatin response.

Dictyostelium discoideum is a species of free-living soil amoeba that feeds on bacteria that grow on decaying vegetation. Though the present account deals with D. discoideum, I use the more colloquial 'dictyostelium' in this article. In 1989, as a new PI, I began to study the response of D. discoideum amoebae to pisatin. Pisatin is the major phytoalexin of the pea plant (Pisum sativum). Phytoalexins are antifungal compounds made by plants in response to infection and injury. No other group has studied any dictyostelium vis-a`-vis any phytoalexin. Evidence for saying so comes from PubMed: four papers show up with the keywords 'dictyostelium', and 'phytoalexin', all from my lab. Why did we 'plough this lonely furrow' and what did we uncover?

A cross-eyed geneticist's view VI. Segregation distortion in Drosophila melanogaster: Recent progress in solving 'an esoteric puzzle'.

Segregation distortion refers to an unusual genetic phenomenon in diploid organisms by which the two alleles at a locus in a parent are not recovered in the classical 1:1 Mendelian ratio in its offspring. The Drosophila melanogaster neogene Sd was created by a duplication breakpoint on the left arm of chromosome 2 (2L), and encodes a truncated RanGAP protein with normal GTPase activity but which mis-localizes to the nucleus and disrupts Ran gradients. Male flies carrying Sd exhibit segregation distortion for the Rsp locus on the right arm of chromosome 2 (2R). Specifically, spermatids inheriting chromosome 2 with the Sd+ Rsps(s) genotype, in heterozygous males of genotype Sd Rspi/ Sd+ Rsps(s), fail to develop properly so that the majority of progeny (approaching 100%) carry just the Sd Rspichromosome. One recent paper reported novel RNAi-expressing transgenes with Sd-mimicking properties; and another reported localization of an X-linked suppressor which restores Mendelian transmission. This article highlights how Drosophila genetics resources made this possible, and the significance of these findings to nucleus-cytoplasm transactions of interest to the wider cell biology community.

Fungal senescence induced by the Neurospora sen mutation and mitochondrial plasmids - the contributions of Ramesh Maheshwari.

This article describes some of the research contributions made by Prof. Ramesh Maheshwari and his colleagues at the Indian Institute of Science, Bangalore. These include (1) the understanding of the Neurospora life cycle in agricultural (sugarcane) fields, (2) identification of Neurospora mutants that trigger vegetative spore development via microcycle conidiation, and (3) isolation of wild Neurospora strains in which the essential immortality of the fungal mycelia is subverted.

A transmission ratio distortion and the 'max-4' ascus phenotype: Do both reflect the same Bateson-Dobzhansky-Muller Incompatibility emerging during trans-species introgression of translocations in Neurospora?

The T(EB4)Nta, T(IBj5)Nta, and T(B362i)NtA strains were constructed by introgressing the insertional translocations EB4, IBj5, and B362i from Neurospora crassa into the related species N. tetrasperma. The progeny from crosses of T(IBj5)Nta and T(B362i)NtA with opposite mating-type derivatives of the standard N. tetrasperma strain 85 exhibited a unique and unprecedented transmission ratio distortion (TRD) that disfavored homokaryons produced following alternate segregation relative to those produced following adjacent-1 segregation. The TRD was not evident among the [mat A + mat a] dikaryons produced following either segregation. Further, crosses of the T(IBj5)Nta and T(B362i)NtA strains with the Eight spore (E) mutant showed an unusual ascus phenotype called 'max-4'. We propose that the TRD and the max-4 phenotype are manifestations of the same Bateson-Dobzhansky-Muller incompatibility (BDMI). Since the TRD selects against 2/3 of the homokaryotic progeny from each introgression cross, the BDMI would have enriched for the dikaryotic progeny in the viable ascospores, and thus, paradoxically, facilitated the introgressions.

The Neurospora crassa Standard Oak Ridge Background Exhibits Atypically Efficient Meiotic Silencing by Unpaired DNA.

Meiotic silencing by unpaired DNA (MSUD), an RNAi-mediated gene silencing process, is efficient in crosses made in the Neurospora crassa standard Oak Ridge (OR) genetic background. However, MSUD was decidedly less efficient when the OR-derived MSUD testers were crossed with many wild-isolated strains (W), suggesting that either sequence heterozygosity in tester x W crosses suppresses MSUD, or that OR represents the MSUD-conducive extreme in the range of genetic variation in MSUD efficiency. Our results support the latter model. MSUD was less efficient in near-isogenic crosses made in the novel N. crassa B/S1 genetic background, and in N. tetrasperma strain 85. Possibly, in B/S1 and 85, additional regulatory cues, absent from OR, calibrate the MSUD response. A locus in distal chromosome 1R appears to underlie the OR vs. B/S1 difference. Repeat-induced point mutation (RIP) destroys duplicated genes by G:C to A:T mutation of duplicated DNA sequences. Chromosome segment duplications (Dps) dominantly suppress RIP, possibly by titrating out the RIP machinery. In Dp x N crosses, the Dp-borne genes cannot pair properly, hence efficient MSUD, as in OR, silences them and renders the crosses barren. We speculate that the increased productivity engendered by inefficient MSUD enables small duplications to escape RIP.

A cross-eyed geneticist's view I. Making sense of the lamin B receptor, a chimeric protein.

The vertebrate inner nuclear membrane protein, lamin B receptor, has an N-terminal ~200 residue nucleoplasmic domain (NTD), and a ~420 residue C-terminal domain (CTD) that anchors the NTD to the INM. Chen et al (2016) showed the NTD interacts with Xist long noncoding RNA to effect X chromosome inactivation in female mammals. Tsai et al (2016) showed the CTD has sterol reductase activity that is essential for viability. And Nikolakaki et al (2017) proposed a model to interconnect these disparate functions of this chimeric protein. It amuses me now to think back to 24 years ago, when I was concerned that these domains might have come together in a cloning artifact.

Ascus dysgenesis in hybrid crosses of Neurospora and Sordaria (Sordariaceae).

When two lineages derived from a common ancestor become reproductively isolated (e.g. Neurospora crassa and N. tetrasperma), genes that have undergone mutation and adaptive evolution in one lineage can potentially become dysfunctional when transferred into the other, since other genes have undergone mutation and evolution in the second lineage, and the derived alleles were never 'tested' together before hybrid formation. Bateson (1909), Dobzhansky (1936), and Muller (1942) recognized that incompatibility between the derived alleles could potentially make the hybrid lethal, sterile, or display some other detriment. Alternatively, the detrimental effects seen in crosses with the hybrids may result from the silencing of ascus-development genes by meiotic silencing by unpaired DNA (MSUD). Aberrant transcripts from genes improperly paired in meiosis are processed into single-stranded MSUD-associated small interfering RNA (masiRNA), which is used to degrade complementary mRNA. Recently, backcrosses of N. crassa / N. tetrasperma hybrid translocation strains with wild-type N. tetrasperma were found to elicit novel ascus dysgenesis phenotypes. One was a transmission ratio distortion that apparently disfavoured the homokaryotic ascospores formed following alternate segregation. Another was the production of heterokaryotic ascospores in eight-spored asci. Lewis (1969) also had reported sighting rare eight-spored asci with heterokaryotic ascospores in interspecific crosses in Sordaria, a related genus. Ordinarily, in both Neurospora and Sordaria, the ascospores are partitioned at the eight-nucleus stage, and ascospores in eight-spored asci are initially uninucleate. Evidently, in hybrid crosses of the family Sordariaceae, ascospore partitioning can be delayed until after one or more mitoses following the postmeiotic mitosis.

Neurospora tetrasperma crosses heterozygous for hybrid translocation strains produce rare eight-spored asci-bearing heterokaryotic ascospores.

During ascogenesis in Neurospora, the ascospores are partitioned at the eight-nucleus stage that follows meiosis and a post-meiotic mitosis, and the ascospores that form in eight-spored asci are usually homokaryotic. We had previously created novel TNt strains by introgressing four Neurospora crassa insertional translocations (EB4, IBj5, UK14-1, and B362i) into N. tetrasperma. We now show that crosses of all the TNt strains with single-mating-type derivatives of the standard N. tetrasperma pseudohomothallic strain 85 (viz. TNta x 85A or TNtA x 85a) can produce rare eight-spored asci that contain heterokaryotic ascospores, or ascospores with other unexpected genotypes. Our results suggest that these rare asci result from the interposition of additional mitoses between the post-meiotic mitosis and the partitioning of nuclei into ascospores, leading to the formation of supernumerary nuclei that then generate the heterokaryotic ascospores. The rare asci probably represent a background level of ascus dysgenesis wherein the partitioning of ascospores becomes uncoupled from the post-meiotic mitosis. Ordinarily, the severest effect of such dysgenesis, the production of mating-type heterokaryons, would be suppressed by the N. crassa tol (tolerant) gene, thus explaining why such dysgenesis remained undetected thus far.

Crosses Heterozygous for Hybrid Neurospora Translocation Strains Show Transmission Ratio Distortion Disfavoring Homokaryotic Ascospores Made Following Alternate Segregation.

By introgressing Neurospora crassa translocations into N. tetrasperma, we constructed heterokaryons bearing haploid nuclei of opposite mating types, and either the translocation and normal sequence chromosomes (i.e., [T + N]) or a duplication and its complementary deficiency (i.e., [Dp + Df]). The [T + N] heterokaryons result from alternate segregation of homologous centromeres, whereas adjacent-1 segregation generates [Dp + Df]. Self-cross of either heterokaryon produces [T + N] and [Dp + Df] progeny. Occasionally during N. tetrasperma ascus development, a pair of smaller homokaryotic ascospores replaces a heterokaryotic ascospore. Crosses with the Eight-spore mutant increase such replacement, and can generate asci with eight homokaryotic ascospores, either 4T + 4N from alternate segregation, or 4Dp + 4Df from adjacent-1 segregation. Crosses of some of the introgressed translocation strains with normal sequence N. tetrasperma produced more Dp than T or N homokaryotic progeny. We suggest this is due to an insufficiency for a presumptive ascospore maturation factor, which increases the chance that, in asci with > 4 viable ascospores, none properly mature. Since only four viable ascospores (Dp or [Dp + Df]) share the limiting factor following adjacent-1 segregation, whereas four to eight ascospores compete for it following alternate segregation, this would explain why Dp homokaryons outnumber T and N types, whereas the heterokaryons are not as affected. We believe that this novel form of transmission ratio distortion is caused by a Bateson-Dobzhansky-Muller Incompatibility (BDMI) triggered by an N. crassa gene in the N. tetrasperma background. Heterokaryons tend not to out-cross, and crosses of Dp strains are barren, thus the BDMI impedes interspecies gene flow.

Neurospora Heterokaryons with Complementary Duplications and Deficiencies in Their Constituent Nuclei Provide an Approach to Identify Nucleus-Limited Genes.

Introgression is the transfer of genes or genomic regions from one species into another via hybridization and back-crosses. We have introgressed four translocations (EB4, IBj5, UK14-1, and B362i) from Neurospora crassa into N. tetrasperma. This enabled us to construct two general types of heterokaryons with mat-A and mat-a nuclei of different genotypes: one type is [T + N] (with one translocation nucleus and one normal sequence nucleus), and the other is [Dp + Df] (with one nucleus carrying a duplication of the translocation region and the other being deleted for the translocation region). Self-crossing these heterokaryons again produced [T + N] and [Dp + Df] progeny. From conidia (vegetative spores) produced by the heterokaryotic mycelia, we obtained self-fertile (heterokaryotic) and self-sterile (homokaryotic) derivative strains. [T + N] heterokaryons produced homokaryotic conidial derivatives of both mating types, but [Dp + Df] heterokaryons produced viable conidial homokaryons of only the mating type of the Dp nucleus. All four [T + N] heterokaryons and three [Dp + Df] heterokaryons produced both self-sterile and self-fertile conidial derivatives, but the [Dp(B362i) + Df(B362i)] heterokaryons produced only self-sterile ones. Conceivably, the Df(B362i) nuclei may be deleted for a nucleus-limited gene required for efficient mitosis or nuclear division, and whose deficit is not complemented by the neighboring Dp(B362i) nuclei. A cross involving Dp(EB4) showed repeat-induced point mutation (RIP). Because RIP can occur in self-crosses of [Dp + Df] but not [T + N] heterokaryons, RIP alteration of a translocated segment would depend on the relative numbers of [Dp + Df] vs. [T + N] ancestors.