The use of model organisms to understand essential processes is a well-known strategy in life science research. If we take a look at the publication statistics in repositories like PubMed, we can see that there are a substantial number of studies using different model organisms. For example, a quick search in the aforementioned repository resulted in (accessed on 2023.10.16) 142,277 matches for the keywords ‘Saccharomyces cerevisiae’, 429,254 for ‘Escherichia coli’, 1,945,515 for ‘Mus musculus’, 92,105 for ‘Arabidopsis thaliana’, 62,541 for ‘Drosophila melanogaster’, 36,775 for ‘Caenorhabditis elegans’ just a few to mention. This leads us to conclude that the contribution of model organisms to our understanding of basic biological processes is indispensable.

The yeasts have a special place among model organisms because these tiny fungal cells have provided many useful models for different studies. For example, Candida albicans and Cryptococcus neoformans emerged as models for studying fungal pathogenesis, while S. cerevisiae and Schizosaccharomyces pombe are useful models for studying the eukaryotic cell cycle and other countless fundamental biological processes. Accordingly, thousands of research articles related to these species are published every year (Fig. 1). Although S. cerevisiae is the most popular yeast model, we would like to concentrate on the fission yeasts (Schizosaccharomyces) as models in this particular review. The fission yeasts are widely established models of the eukaryotic cell cycle, cell size maintenance, cellular aging, gene expression and epigenetics, autophagy, and apoptotic processes, just a few to mention.

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FIGURE 1: Number of published papers at the PubMed repository from 2020 to 2023. S. cerevisiae is for the baker’s yeast Saccharomyces cerevisiae, and C. albicans stands for the opportunistic human pathogen Candida albicans. C. neoformans represents the medically important species Cryptococcus neoformans, and S. pombe is for the fission yeast model Schizosaccharomyces pombe.

To our best knowledge, the fission yeast genus consists of six species to date: S. japonicus, S. pombe, S. octosporus, S. cryophilus, and the recently described species S. osmophilus and S. lindneri, and other variants 1, 2, 3, 4, 5, 6. S. japonicus has two main varieties: var. japonicus and var. versatilis, which have recently been proposed to be considered as two different lineages 7. In our opinion, the two most divergent branches of the genus (S. japonicus and S. pombe) have tremendous potential (not just) as model organisms.

FISSION YEASTS? FOR WHAT?

Someone may ask the legitimate question: why do we need fission yeasts when we already have well-established and widely used yeast models such as S. cerevisiae and C. albicans? What can fission yeasts provide that cannot be provided by the aforementioned ones? Most importantly, could a fission yeast be a good (or better) alternative for studying human diseases than budding yeasts? We try to provide answers to these questions while revealing some fundamental differences among the yeast models (Table 1).

THE ADVANTAGES OF FISSION YEASTS IN VIRUS RESEARCH

Viruses can cause various and often fatal diseases. Effective prevention and treatment of these diseases require extensive knowledge about the molecular mechanism of the infection and the changes caused by the viral proteins in the host cells. Various model organisms are used as hosts to reveal consequences of viral infections. The yeasts belong to these model organisms 115, because of their attractive features, such as eukaryotic cell structure, small genome, widely available molecular tools, and the ability of several eukaryotic viruses to replicate in their cells 20, 116, 117. That is, yeast cells are suitable for heterologous expression and the study of viral proteins.

Here, we would like to provide a brief insight into the research results of viral proteins produced in the fission yeast S. pombe, with particular attention to the Human Immunodeficiency Virus type 1, (HIV-1) Vpr protein, which has been extensively studied in this yeast species.

HIV1 causes Acquired Immunodeficiency Syndrome (AIDS) by damaging the immune system, which is a life-threatening condition. The HIV-1 genome contains several genes, and each protein encoded by these genes has a special role 118, 119. Cloning of these viral genes into S. pombe-specific vectors allowed the researchers to determine the exact cellular localization of the GFP (Green Fluorescent Protein)-tagged viral proteins in the yeast cells 119. The localization of many proteins was revealed for the first time, while further results demonstrated that the intracellular localization of the viral proteins was the same in the yeast and human cells 119.

The Vpr gene (Virus protein R) which encodes a component of virus particles that promotes virus infectivity, has been studied in detail 118. One of the goals was to find out which cellular processes of the host cells are affected by the Vpr protein and whether the same processes are inhibited in the yeast cells and the human cells. Since the S. pombe genomic sequence 14, and the genetic background of its cell processes were well-known, and in addition, a large number of mutant strains were available in this species, it was possible to express the Vpr gene both in the wild-type and various mutant strains. The overproduction of the Vpr gene product revealed that the Vpr protein caused multiple effects on the host cells. The expression of the viral protein resulted in small colonies, growth delay, abnormal cell morphology, arrest in the G2 phase of the cell cycle, and cell death 120, 121, 122, 123, 124. Besides, the Vpr protein caused depletion of the glutathione, and oxidative stress, stimulating the production of reactive oxygen species (ROS) 124, 125, 126. In addition, the direct interaction of the Vpr protein with the proteosome complex, which is responsible for ubiquitin-mediated protein degradation, has also been demonstrated (Fig. 2) 127.

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FIGURE 2: The Vpr protein caused multiple effects on the S. pombe cells.

To find out how a single viral protein can destroy various cellular processes, the phenotypic changes of the transformed yeast cells were investigated. Examination of cell morphology of the Vpr-transformed cells showed that the changes were caused by several cellular abnormalities, such as disruption of actin cytoskeleton or altered cell polarity 121. Cloning and transformation of the mutant Vpr genes enabled the detection of the effect of a given mutation on the Vpr function. The results obtained in S. pombe showed highly similar changes to the human cells that confirmed the conservation of the Vpr functions. Besides, the truncated genes also revealed that the C-terminal end of the Vpr protein was particularly important for the cell cycle (G2) arrest, while the N-terminal region was required for nuclear localization 128. Chen’s report also demonstrated that the nuclear localization of the Vpr protein was not required for G2 arrest, while it was necessary for cell killing, suggesting that the G2 arrest and cell death caused by Vpr could be independent functions 128.

The investigation of the Vpr-expressing yeast cells shed also light on the molecular background of the cell cycle arrest. The experiments proved that cell cycle arrest correlated well with increased phosphorylation of the Cdc2 kinase, which is the key regulator of mitosis 120, 129. These experiments showed that the regulators of the Cdc2, such as wee1 (encodes an M-phase inhibitor protein kinase) and the cdc25 (encodes a phosphatase, M-phase inducer) were important in the Vpr-induced cell cycle arrest 130, 131. According to the data, the Vpr protein promoted the cytoplasmic compartmentalization of Cdc25 and inhibited its function, which required the Srk1 kinase 123. Since there are differences in cell cycle regulation between S. pombe and S. cerevisiae (the G2/M transition is more important in S. pombe than S. cerevisiae) 53, it was better to choose the fission yeast for the analysis of Vpr-mitosis relation.

The further results also showed that the Vpr protein might affect the cell cycle through different pathways because the rad24 gene (which plays a role in the DNA damage pathway) was also involved in the Vpr-associated cell division defect 131. Based on these results a putative mechanism of the Vpr-induced cell cycle arrest could also be determined 131. The genetic screens, where checkpoint and mitotic regulator mutants were used, have confirmed the complexity of the viral effect, and shed light on the role of further genes, such as rad25, wos2, and hsp16, ef2 that enhanced or suppressed the cell cycle defect or cell death caused by the Vpr protein 124, 130, 132, 133. Examination of Vpr-induced cell death demonstrated that it resembles apoptosis and correlates with changes in mitochondrial morphology. This study described well the pro-apoptotic effects of Vpr 123.

The S. pombe cells were also suitable for finding agents that can reduce the negative effects of the Vpr protein. The H₂O₂ treatment for example promoted the survival of the Vpr-expressing yeast cells 134, while a simple fission yeast-based screening system allowed to find small molecules that specifically inhibit HIV-1 Vpr 135.

In summary, this simple model organism allowed researchers to reveal the effects of the multifunctional Vpr protein on the host cells. The researchers were able to discover the cellular processes disturbed by the viral protein and their molecular background, while a comparison with the results obtained in mammalian cells showed the conserved characteristics of the viral infection. These results could contribute to a better understanding of the mechanism of viral infection and HIV-1 pathogenesis. Although S. cerevisiae is also used as a model to study the HIV-1 Vpr effects 136, 137, there are some major differences that make S. pombe superior to S. cerevisiae in this regard. Besides the aforementioned cell cycle control point, the Vpr-induced changes in mitochondrial morphology more closely resemble those observed in human cells compared to S. cerevisiae. S. pombe exhibits a greater degree of similarity to humans with respect to mitochondrial features 138. S. pombe displays cell death induced by Vpr that shares some characteristics with apoptosis in human cells, potentially making it a more relevant model for studying this aspect 123.

POMBE FOR PRION BIOLOGY?

Prions are amyloid forms of cellular proteins and are implicated in many incurable and fatal neurodegenerative disorders. Prion disease can be transmitted from organism to organism and is characterized by the accumulation of PrP^Sc (scrapie isoform of the prion protein). The disease has many forms, such as genetic, sporadic, and acquired 139.

Prions seem to be more widespread than currently appreciated because the research data revealed that yeasts can also have heritable elements transmitted via proteins 140, 141. Since many yeast genome sequences are available, they allow the in silico identification of prion-like genes/proteins in different species 142, 143. In this way, genes with various functions, such as transcriptional regulators, genes involved in sporulation, copper-transport, and translation were identified as prion-associated proteins 141, 143. Structural analyses also showed that asparagine/glutamine-rich domains are linked to amyloidogenesis 140.

In S. pombe 295 PrD (prion-forming domain) containing proteins were identified 143 . One of the prion-like proteins is encoded by the ctr4 gene, the study of which, placed S. pombe on the prion map 143, 144. The overexpressed form of this copper transporter protein was proteinase K-resistant and conferred sensitivity to oxidative stress 143. In addition, overexpression of a S. cerevisiae gene (ScSup35) in S. pombe also demonstrated that this fission yeast can support the formation and propagation of the S. cerevisiae prion 143. Experimental examination of the other genes mentioned above may lead to many new results.

Further characterization of chaperons and heat shock proteins (HSP), as the latter genes are linked to protein folding 139, may reveal especially the new details of prion aggregation. A study has revealed for example that the C-terminal region of HSP104 plays an essential role in prion propagation 145, while the results of Reidy and co-workers confirmed the role of other chaperons in prion propagation 146. As also S. pombe has many hsp genes and genes with GO term “heat shock protein binding” (GO:0031072) (PomBase), their investigation can significantly expand our knowledge of prion disease.

THE DARK HORSE OF EUKARYOTIC CELL RESEARCH: SCHIZOSACCHAROMYCES JAPONICUS

The most divergent branch of the fission yeast genus is the dimorphic S. japonicus 44, 10, 147, which has several features that make it an interesting prospect among other model organisms 32, 148, 149.

First and foremost, S. japonicus is able to switch between a unicellular yeast form and a true invasive hyphal form 150, 151, 152, 153, 154. Hyphal switching can occur through different stimuli: nutrient deprivation 150, DNA damage 153, 154, the presence of fetal bovine serum (FBS) or fruit extracts 155, 156, and negatively regulated by quorum sensing 157. S. japonicus is not pathogenic to humans, despite its ability to form invasive hyphae that penetrate solid surfaces like agar or gelatine 150, 156, 158. Moreover, hyphal extension is initiated in the presence of FBS even in liquid media, and elevated transcription levels of certain protease-coding genes can be observed in the hyphae 155, 159. Thus, it can be a good non-pathogenic model to study the fungal dimorphism. However, some unique features distinguish it from other dimorphic species such as C. albicans. S. japonicus hyphae does not have a Spitzenkörper, undergoes complete cell divisions, and remains mononuclear 156. Additionally, one of the master regulators of the yeast-to mycelia transition, the transcription factor Nrg1 behaves differently in S. japonicus. In C. albicans, NRG1 represses morphological transition 160, 161, while in S. japonicus, it rather acts as an activator of the hyphal switch 157, 159. Further differences can be observed as nitrogen starvation is a signal that induces a morphological switch in C. albicans, but it is not effective in S. japonicus 162, 163. In this regard, it seems that the MAPK signal transduction pathways contribute somewhat differently to hyphal induction in S. japonicus than in C. albicans 157, 163. Besides, the hyphae of S. japonicus are photoresponsive, which is also an unusual feature among most of the other yeasts 164.

However, S. japonicus is also quite different from its closest relative. While a handful of studies suggest S. pombe can produce adhesive and invasive hyphae-like phenotypes under specific conditions or certain genetic backgrounds 165, 166, 167, 168, 169, 170, S. japonicus remains the definitive dimorphic species within the genus. Furthermore, S. japonicus utilizes a semi-open form of mitosis, while S. pombe undergoes closed mitosis, they differ in the regulation of chromatin-nuclear envelope interactions during mitosis, moreover they exhibit discrepancies in their dynamics of cytokinesis and gene regulation too 171, 172, 38, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186. For example, while S. pombe assembles the actomyosin ring in metaphase and requires a mechanism to prevent its premature constriction, S. japonicus initiates ring assembly only at the mitotic exit, similarly to metazoan cells 149, 176, 187. Although all the fission yeasts have large, centromeric regions with repetitive sequences, S. japonicus does not have specialized pericentromeric repeat sequences as S. pombe has, but it has a larger complement of retrotransposons clustered at centromeric and telomeric regions 10, 60, 188. The S. japonicus centromeres consist of arrays of retrotransposons, which is reminiscent of the human centromeric structure, moreover, the RNAi pathway is indispensable for both S. japonicus and mammalians 188, 189, 190, 191. In S. japonicus, RNAi-mediated silencing of retrotransposons is essential to maintain centromere function and genome integrity, while the other fission yeasts rely on the CENP-B proteins and use RNAi exclusively for heterochromatin maintenance 10, 60, 188, 192. They exhibit discrepancy in their cell-wall composition too: the O-glycans on the cell surface of S. pombe, S. octosporus, and S. cryophilus mainly composed of tetra-saccharides, whereas those of S. japonicus mostly consist of trisaccharides (Gal-Man-Man) 193, 194. Besides, S. japonicus has a wider temperature tolerance: the growth of S. pombe is largely restricted above 37°C, S. japonicus can even grow at 42°C and the generation time is somewhat shorter of S. japonicus than that of S. pombe 32, 148. Strikingly, S. japonicus is well-adapted to anaerobic conditions as it has respiratory deficiency and is able to grow anaerobically without sterol supplementation, which is an unusual ability among eukaryotic organisms 195, 196, 197, 198. In this context, S. japonicus can grow much faster under fermentative conditions than S. pombe, and produces ethanol even at 42°C 197. Alam and co-workers showed that in spite of the fact that S. japonicus does not respire oxygen, it is capable of efficient NADH oxidation, amino acid synthesis, and ATP generation via modification of metabolic pathways 199. S. japonicus is also a suitable model to study membrane bilayer properties and dynamics in anoxic environments, knowing that numerous changes can occur in the membrane lipidomes under hypoxic conditions, for example, in a tumor microenvironment 200, 201, 202.

The phylogenetic distance within the Schizosaccharomyces genus is uniquely large, despite the fact, that they possess remarkably conserved gene content, gene order and gene structure. According to Sipiczki and Rhind et al., at the level of protein sequence identity (~55%), S. japonicus is as distant from S. pombe as the platypus is to humans 44, 10. Interestingly, there might be no genus of Ascomycota that exhibits such a high degree of gene content conservation and sequence divergence at the same time 48, 49, 203. Such sequence divergence, besides the high amount of common gene content, really provides an excellent model pair to study the same cellular processes in different genetic backgrounds. Since most of the laboratory protocols developed for S. pombe can also be used (with slight modifications) for S. japonicus, the parallel investigation of these two species provides an unprecedented opportunity 174, 201, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215.

After we showed that S. japonicus is a remarkable model organism by itself or in comparison with other species, we can ask the question: what is S. japonicus able to bring to human disease research? The answer is not so trivial.

S. JAPONICUS HYPHAL GROWTH AS A MODEL TO STUDY THE INVADOPODIA OF TUMOR CELLS

Besides the above-mentioned advantages of S. japonicus, so far no comparisons have been made between mammalian cells and hyphal growth. This is not surprising because mammalian cells do not form structures such as hyphae, do they? We can say that mammalian cells do not have structures corresponding to hyphae, except for one that resembles its behavior: the invadopodium.

Generally, invadopodia can be described as membrane protrusions, which play a key role in cancer metastasis. These actin-rich structures can reach a diameter of 3 µm and extend several micrometers in length 216. It can digest the surrounding tissues by proteases, to help disseminate the cancerous cells.

One could say that both invadopodia and hyphae are very specialized structures with different roles. However, invasive cell growth may have a deep origin, in which particular features are common among the different lineages 217. Thus, comparisons can be made along five main aspects: polarized growth, actin cytoskeleton, vesicle trafficking, substrate degradation, and environmental sensing (Fig. 3).

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FIGURE 3: Common features of tumor invadopodia and hyphal growth. (A) Tumor invadopodium invades the extracellular matrix (ECM) among healthy cells. (B) Extension of invasive hypha of the fission yeast S. japonicus in the solid medium among normal vegetative yeast cells. (C) Common features enable a direct comparison between cancer invadopodia (left side) and the fungal hyphae (right side). Polarized growth: both fungal hyphae and invadopodia grow in a polarized way. Actin cytoskeleton: the polarized growth is primarily driven by actin polymerization, and it needs changes in the cytoskeletal structure. Vesicle trafficking: invadopodia formation or hyphae growth is unimaginable without vesicle transport. Matrix/substrate degradation: to continue expansion and acquire nutrition, both the invadopodia and the hyphae need to release enzymes that degrade their surrounding environment. Environmental sensing: signals from the environment have a substantial impact on the behavior of cells. Invadopodia formation and yeast-to-hyphae transition are affected by environmental factors like nutrient availability, pH, temperature, or CO2. *Although transcriptome analysis of the hyphae of S. japonicus suggested that several coding genes responsible for the production of vacuolar hydrolases were upregulated during hyphal extension [159], further studies are required to assess the extent of substrate degradation in S. japonicus.

Common orthologues and gene regulation

As we have seen in these subchapters, yeast-to-hyphae transition and invadopodia formation have many features in common and are comparable to each other. Despite their distinct functionality, the core mechanisms are very similar. To determine whether these two processes share common genes, we compared the RNA sequencing data from S. japonicus hyphae and invadopodia, without claiming completeness 159, 243. In the case of the S. japonicus hyphae, 1337 genes were significantly upregulated, of which 112 genes showed expression above log₂ fold change 2 159. 1484 genes were significantly downregulated, among which 109 had log₂ fold changes below 2 159. In the cancer invadopodia, 5873 genes showed elevated expression, while 5467 genes showed decreased expression levels 243. Based on the data of JaponicusDB (accessed on 2024.07.02.), S. japonicus and humans share ~3500 common orthologues (https://www.japonicusdb.org/data/orthologs/). According to our more stringent approach, strict reciprocal BLASTp analyses (E value ≤ 1 × 10^-30) revealed 1774 common putative orthologues between S. japonicus and H. sapiens (Supplementary Table S2) 16. The list of common orthologues was compared to the gene lists of the RNA seq obtained from S. japonicus hyphae 159 and human invadopodia 243, which resulted in a total of 85 common genes (Supplementary Table S3). These genes were subjected to categorization by Gene Ontology (GO) terms, considering mainly the common biological processes. In this way, the common orthologue number was reduced to 53 (Supplementary Table S4). 26 genes out of 53 exhibited similar regulation in both hyphae and invadopodia (Table 2), whereas 27 genes showed opposite regulation (Supplementary Table S4). Several gene pairs belonged to the GO categories of transport or metabolic processes (Table 2 and Supplementary table S4). Although the number of common orthologues is quite small and half of the genes were differently regulated, they might still be good indicators or starting points for further investigation of regulatory mechanisms.

 

We should bear in mind that this is only one pairwise comparison from one-on-one specific conditions, data from different circumstances might result in substantially different gene sets. Moreover, we should also consider the fact that invadopodia are formed in cells that carry severe genetic mutations, in contrast to the normal genetic background of S. japonicus in which the hyphae were produced. Taking these considerations into account, we would consider it particularly interesting to examine the S. japonicus hyphae production in such a mutational genetic background that resembles the genetic background of the invadopodia. In particular because some S. japonicus cell cycle mutant strains produced somewhat different hyphae (Fig. 4 C, D), compared to the wild-type strain (Fig. 4 A, B).

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FIGURE 4: Microscopic and macroscopic morphologies of different Schizosaccharomyces japonicus strains. Microscopic morphology of the wild-type S. japonicus cells (A) and colony morphology of the yeast phase and hyphae on agar plate (indicated with white arrows and labels) (B). Cell morphology of a yet unidentified cell separation mutant strain (C) and its yeast phase and hyphae production (D). Cell sizes in (A) and (C) are not to scale, the images concentrate on the cell morphology. Scale bars represent 10 µm. Microscopic images were captured with an Olympus DX-40 microscope and an Olympus DP-70 camera. Photos were taken in different focal planes and stacked with the program Combine ZP.

LABORATORY USE AND TOOLKITS

Although there is a tendency for most molecular toolkits to be developed for S. cerevisiae first, then adapt to the fission yeasts, sometimes the latter species proves to be a better subject in terms of practical implications. Many wild-type S. cerevisiae strains are used as laboratory models, and because of this, it often happens that the same mutation causes different phenotypes in different wild-type strains 244, 245. This often makes the comparison of the results difficult. In contrast, almost every lab working with S. pombe uses the same strains: the L968 h⁹⁰ homotallic, the L972 h^– and the L975 h⁺ heterotallic strains isolated by Urs Leupold 246. As a result, majority of the studies using S. pombe can be directly compared and contrasted. Furthermore, L968 is a natural isolate, which does not behave differently compared to the other natural isolates 247.

Numerous useful databases, protocols and toolkits have arisen through the years for the fission yeasts. Virtually, all the molecular biology tools available were adapted or can be adapted to fission yeasts, from standard gene replacements to CRISPR-Cas9 and from FISH to Hi-C systems 248, 249, 250, 251, 252. Since Herrera-Camacho and co-workers have presented many useful applications for S. pombe, here we just focus on the recently described methods, online tools and algorithms (Table 3 and 4) 251.

Both S. pombe and S. japonicus have their own dedicated databases: PomBase and JaponicusDB, which are community-curated (Table 3) 14, 15. These platforms summarize the results reported by the researchers working with the fission yeasts; they enable a rapid overview of the recent developments in many topics.

LIMITATIONS

Despite all good features of yeasts, they also have their own limiting factors. Since all the fission yeast species have their unique elements of metabolic pathways and protein interaction networks, most of the biological processes can only be “similar” to their human counterparts. Although we could gain useful information about human diseases using fission yeast models 297, 298, 299, 300, 301, 302, 303, 304, 305, there will always be differences that we should be cautious about. At the same time, complex processes cannot be investigated because of the lack of multicellular phenotypes. But beyond the trivial, there are other factors to consider.

The creation of auxotrophic mutant strains is a widely used procedure in yeast genetics. Auxotrophic mutant strains enable researchers to easily verify the success of a gene deletion or plasmid vector introduction into the cells, for example. However, there is emerging evidence that a knockdown of even a simple metabolic gene could produce a pleiotropic effect, which causes a complex phenotype leading to false conclusions. For instance, a defect in certain amino acid (AA) biosynthetic pathways may activate the general AA control and suppress the TOR pathway, depending on the growth conditions 306, 307, 308. In the case of S. pombe, leucine (Leu) auxotroph strains have been used for decades 309, although Leu auxotrophy can cause altered intracellular response compared to the prototrophic wild-type strain 307. Similarly, the use of the URA3 gene as a selective marker caused decreased virulence in C. albicans, thus, it resulted in misleading phenotypes 310, 311. The effect could be more severe in the case of strains that have two or more auxotrophies. The situation is not much better when using antibiotic-resistant genes as genetic markers. To ensure a sufficient expression of the marker gene, constitutive promoters are generally used. Those promoters sometimes act bidirectional or might elevate the expression levels of the neighboring genes too 312. In that particular case, we are again facing a pleiotropic phenotype. It is also common practice to knock out one of the members of the non-homologous DNA end joining (NHEJ) repair system to enhance gene targeting efficiency. Without efficient NHEJ, the cells ideally use the homologous recombination repair system, which enables precise integration of the foreign DNA into the target genome. Despite NHEJ-deficient strains showing normal phenotypes in standard circumstances, NHEJ members have many other roles that go beyond just joining DNA ends. In S. pombe, the Pku70-Pku80 heterodimer plays a critical role in telomere length maintenance and recovery from replication stress 313, 314. In S. japonicus, disruption of the Ligase 4 (lig4) gene resulted in a seemingly normal phenotype 315. However, increased sporulation on complete medium, decreased hyphal growth, faster chronological aging, and higher sensitivity to heat shock, UV light, and caffeine can be observed in the lig4-disrupted strains 315. Thus, gene characterization conducted in an NHEJ-deficient strain could also lead to incorrect conclusions.

Although we are able to track dynamic cellular processes by using chemical inhibitors, many drugs are not efficient enough for fission yeasts because of their multidrug resistance (MDR) 316, 317, 318. Thus, finding a new therapeutic agent or investigating the performance of a candidate might not work well in the fission yeast (or in other yeasts). However, there are several counterexamples, of course 319, 320. It is even possible to make fission yeasts sensitive to drugs by engineering their MDR-related genes 318, but the effect of those gene deletions might resemble the gene deletion effects of other metabolic or DNA repair pathway genes.

In fact, any gene knockdown could cause secondary gene mutations or overall genomic imbalance, which initiates adaptive genomic changes 321. Of course, the purpose of many studies is exactly to understand these changes. But we should bear in mind, that all the yeasts have a very short generation time, and the continuous inoculation of their cells could result in multiple bottlenecks and in parallel, forced genome evolution. There is a lot of anecdotal evidence circulating among researchers when they experience that a mutant yeast strain completely changes its behavior after a certain number of rounds of inoculation. To provide experimental evidence, Szamecz et al. showed that many knockout strains have recovered and exhibited almost as good fitness as the wild-type strain did after certain rounds of generation 322. Although they performed their experiments with S. cerevisiae, their result could easily be true for the fission yeasts too.

CONCLUSIONS

With this particular review, we would have liked to emphasize the importance of the fission yeast models, as we are convinced that they still have many unexploited benefits. Although the number of researchers using fission yeasts is relatively high, the size of the fission yeast community is nowhere near the size of the budding yeast community. Obviously, in many cases, it is easier to work with S. cerevisiae, but the fission yeasts share substantially more fundamental biological processes with the metazoans. Therefore, we wanted to highlight some of the differences between budding and fission yeast models, without claiming completeness. Besides, we also wanted to promote S. japonicus as a less-known, but emerging model organism with unique features. As we have shown, S. japonicus is more similar to mammalian cells in certain features than S. pombe is. Moreover, in our view, S. japonicus can easily be a non-mammalian model for tumor invadopodia studies, since the fundamental processes of invadopodia formation and hyphae formation can be rationally compared. Although it is obvious that none of the yeasts can be used as equivalent models of human diseases, we believe that the fission yeasts could substantially contribute to our understanding of the molecular background of human diseases.