Deletion of Dnm1 Gene In Mitochondria Lead To The Changes of Cell Dynamics and Energy Metabolism In Fission Yeast

Mitochondria are dynamic organelles that undergo cycles of ssion and fusion. The major mitochondrial ssion protein is dynamin-related Drp1 GTPase (Dnm1 in yeast). The effects of Dnm1 gene deletion on cell dynamics and energy metabolism during mitosis were studied in Schizosaccharomyces pombe. Dnm1 gene deletion can lead to slow growth, abnormal sporulation, abnormal number and length of interphase microtubules of Schizosaccharomyces pombe. The deletion of Dnm1 gene can also affect the spindle growth speed and growth time of metaphase and anaphase, and affect the spindle uorescence intensity of prophase and metaphase. At the same time, the structure and dynamics of the spindle microtubules of Dnm1Δ are also different. The statistics of spindle length showed that there was delayed spindle fracture in Dnm1Δ Cells. Two different chromosome behaviors, normal and lagged, were observed by living cell imaging. The analysis of coenzymes, intermediates and energy in energy metabolism showed that there were some abnormalities after Dnm1 gene deletion, including coenzyme defects, intermediate metabolite defects and ATP production defects.


Introduction
Mitochondria are dynamic organelles that undergo a cycle of division and fusion (Francy et  The main dynamic related protein of mitochondria is Drp1 GTPase (Dnm1 in yeast) (Naumann et al. 1992; Chang et al. 2010;Bleazard et al. 1999). The structure of GTPase in dynamin family is similar, including GTP binding region, intermediate region and GTPase effect region, but the function is different.
It is reported that the mitochondria in Dnm1Δ present as highly interconnected tubules forming a network structure or thick bundle like tubules, which indicates that Dnm1 is necessary for mitochondrial ssion (Mears et al. 2011). Dnm1 mediates mitochondrial ssion at interphase growth and at cell division during mitosis (Karren et al. 2005). In Saccharomyces cerevisiae, Dnm1p (called DLP1, Drp1, or dvlp1 in other species) is captured by s1p / mdv2p together with net2p / gag3p / mdv1p / s2p and mediates mitochondrial division (Tieu et al. 2002). In addition, the repetitive roles of Dnm1 and vps1 in cell cycle regulated peroxisome biogenesis were also reported (Kuravi et al. 2006).
Cell replication involves a series of highly regulated and evolutionarily conserved complex events, known as "cell cycle" (Syrovatkina et al. 2013). Cell cycle abnormalities have a serious impact and may lead to cancer growth. A detailed understanding of the cell cycle and its regulation can identify other targets for cancer treatment. Schizosaccharomyces pombe ( ssion yeast) is a single celled organism, easy to genetic operation, with many cell cycle characteristics similar to metazoan cells (Dumont et al. 2009). It is an important model organism for studying cell cycle and checkpoint control (Goshima et al 2010). In this paper, the effect of Dnm1 gene deletion on cell mitotic dynamics was studied by using the model of Schizosaccharomyces pombe ( ssion yeast).

S. pombe strains construction
Yeast genetics was carried out as previously described (Forsburg et al. 2006), and yeast strains were created by random spore digestion. Yeast culture media were purchased from For Medium (Norfolk, UK).
All strains used in this study are listed in Table 1.  Living cell imaging was performed at room temperature 25 °C. A spinning-disc confocal microscope with a Nikon PlanApo 100×/1.40 NA objective and the Photometrics CoolSNAP HQ2 CCD camera was used (Tran et al. 2004). MetaMorph 7.5 (http://www.moleculardevices.com) was used to collect and process all images. In order to obtain high temporal resolution, images were acquired at 300-500 ms exposure for GFP/mCherry, 60-sec intervals, 90 min total time for 11 optical sections of 0.5 µm spacing (Rongmei et al. 2020).
Analysis method of energy metabolites The cells were cultured in YE5S medium, 25 °C for 3 days. The cells were washed three times with cold PBS, collected in a 1.5 ml centrifuge tube, frozen in liquid nitrogen, stored at -80 °C, after centrifuged with 3000 g at 4 °C for 5 minutes. The collected cells were sent to Shanghai applied protein Technology Co.
Ltd. to be analyzed for metabonomics by LCMS.

Data analysis
All data were expressed by mean ± SD, and the signi cant difference between the experimental group and the control group was statistically analyzed by one-way ANOVA using SPSS 17 software. * P<0.05, represents a signi cant difference, and ** P<0.01 represents an extremely signi cant difference, respectively.

Results
The effect of Dnm1 gene deletion on the cell growth, morphology and number of microtubule and ascospores The results of cell growth showed that there was no signi cant difference between the wild type and Dnm1Δ Cells at 25 ° C for 0-6 hours. After 6 hours, the growth rate of wild type was faster than that of Dnm1Δ. After 12 hours, the OD 595 of wild type and Dnm1Δ cells reached 0.625 and 0.202, respectively, which is very different from the wild-type cells (Fig. 1A). The results showed that Dnm1 gene deletion could slow down the growth of Schizosaccharomyces pombe. The number of ascospores produced by wild type and Dnm1Δ cells as shown in (Figs. 1B and 1C). The results showed that 99.20 ± 0.00%, 0.53 ± 0.31% and 0.27 ± 0.31% of wild-type cells produce four, three and two ascospores (n = 1500), respectively, while 95.07 ± 0.81%, 4.73 ± 0.64% and 0.00 ± 0.00% of Dnm1Δ cells produce four, three and two ascospores, respectively, in which there had extremely signi cant difference (P < 0.01) in the number of four and three ascospores (Figs. 1B and 1C). There was no signi cant difference in the morphology of ascospores between wild type and Dnm1Δ cells, which showed that Dnm1 gene and its encoded protein had an effect on the production of sporozoites, but had no effect on the morphology of sporozoites.  1D and 1E). The statistical results of microtubule length in mitotic interphase cells showed that microtubule length of wild type and Dnm1Δ cells were 5.13 ± 1.44 µm and 5.97 ± 2.14 µm, respectively, which had extremely signi cant difference in the two groups (Fig. 1F). The results showed that the deletion of Dnm1 gene resulted in the increase of microtubule length. The results of microtubule dynamics of wild type cells and Dnm1Δ cells during mitotic interphase showed that the MT (Microtubule) of wild-type cells grew at 1.50 ± 0.56 µm/min (n = 10) and shrunk at 1.40 ± 0.30 µm/min (n = 10), and the MT dwell time was 1.21 ± 0.21 min (n = 10). In contrast, the MT of Dnm1Δ cells grew at 1.57 ± 0.85 µm/min (n = 10) and shrunk at 1.21 ± 0.47 µm/min (n = 10), and the MT dwell time was 1.16 ± 0.31 min (n = 10) (Figs. 1G-1J). The results indicated that there were no different of microtubule dynamics during mitotic interphase between wild type and Dnm1Δ cells.
The effect of Dnm1 gene deletion on spindle and cell length during cell mitosis In the process of mitosis, SPBs (spindlepoly body) organize the mitotic spindle for chromosome separation. The mitotic spindle has three different elongation stages corresponding to different mitotic stages. The SPBs also organize the astral MTs, and its function is similar to interphase MTs in nuclear and spindle positioning. The mC-Atb2 (α-tubulin) and Hht2-GFP (Nucleosomal histone) were used as a detection signal for prophase and metaphase to explore spindle elongation and chromosome segregation dynamics in cell mitosis of wild-type cells and Dnm1Δ cells. Wild-type cells exhibited typical three-phase spindle elongation kinetics, corresponding to prophase (phase I), metaphase (phase II), and anaphase A (chromatid separation) and B (spindle elongation) ( Fig. 2A)  were no difference between wild type and Dnm1Δ cells, while the statistical results of the uorescence intensity of the spindle at prophase and metaphase showed that the uorescence intensity of the spindle in Dnm1Δ cells were higher than that in the wild type cells (Figs. 3F and 3G). At the same time, the structure and dynamics of the spindle microtubules were different in Dnm1Δ cells compared with wild type. In wild type cells, mitosis and microtubule disintegration occurred simultaneously. In order to standardize the measurement of mitotic time, we de ned the mitotic initiation time of 0 minute as the complete disintegration of cytoplasmic interphase microtubules. In wild type, time 0 min was consistent with the assembly of a microtubule "bar" (82.67 ± 6.63% of cells) or a "dot" (17. . Both values were also signi cantly different between wild type and Dnm1Δ cells, and duration time of anaphase of wild-type were extremely signi cant longer than Dnm1Δ cells, which showed that there was delayed spindle breakage in Dnm1Δ cells. Chromosome segregation is an important cellular process and requires absolute delity, because errors would lead to developmental defects and diseases. The delity of chromosome segregation depends largely on the correct attachment of metaphase kinetochore and MT in metaphase. Chromosome segregation requires the assembly of spindles, which are based on microtubule (MT) structure and can effectively capture and separate sister chromatids during mitosis. The minus end of MT converges to the spindle pole, while the plus end of MT diverges from the opposite pole interdigitation in the middle of spindle. Mutations that alter the length of metaphase stable spindles are associated with chromosome segregation defects. Two different kinds of chromosome behaviors were observed: normal, the chromosome separate to opposite poles at anaphase; lagging, the chromosome is mis-separate to one pole but eventually corrected and separated to opposite poles (Fig. 4D). In Dnm1Δ wild-type cells, the spindle breaked in the form of lineartype (29.33 ± 4.66%), arch-type (41.67 ± 6.41%) and S-type (29.00 ± 5.66%), but in wild-type cells, there were only linear-type and arch-type, and the percentage of the two form were 49.67 ± 4.66% and 53.33 ± 6.44%, respectively (Figs. 4E and 4F). The results showed that the loss of Dnm1 gene could lead to abnormal spindle breakage.
The effect of Dnm1 gene deletion on coenzyme in energy metabolism Tricarboxylic acid cycle, oxidative phosphorylation pathway and glycolysis pathway are the main pathways of cell energy production. In order to further understand the energy metabolism of Dnm1Δ cells, the coenzyme and the energy metabolites in these process were detected by LC-MS. The results showed that the relative contents of avin mononucleotide (FMN  The NADPH production was signi cantly reduced compared with wild-type cells, suggesting that Dnm1 gene deletion affected the hydrogen transfer of NADPH and the oxidative phosphorylation process. At the same time, the production of intermediates of energy metabolism showed abnormal situations in Dnm1Δ cells. The production of D-glucose 6-photosphate, β-D-fructose 6-photosphate, citrate and cis-aconitate, decreased extremely signi cantly (P < 0.01), and pyruvate, isocitrate and L-malic acid decreased signi cantly (P < 0.05). The production of D-glucose 6-phosphate and β-D-Fructose 6-phosphate, which appeared in the speed limiting step of glycolysis (Jojima and Inui 2015), are signi cantly reduced, suggesting that Dnm1 gene deletion affected the key steps of glycolysis and further affects the speed of glycolysis. Pyruvate is the nal product of glycolysis, and is nally imported into mitochondria as the main fuel of the tricarboxylic acid cycle. In mitochondria, pyruvate drives ATP production through a variety of biosynthetic pathways that intersect oxidative phosphorylation and the tricarboxylic acid cycle (Gray et al. 2014). In addition, pyruvate also plays an important role in the metabolism of three major nutrients (Owen et al. 2002). Abnormal metabolism of pyruvate plays an important role in cancer, heart failure and neurodegeneration (Gray et al. 2014). Compared with wild type, pyruvate decreased signi cantly in Dnm1Δ cells, suggesting that Dnm1 gene deletion can reduce the amount of pyruvate and affect ATP production and metabolism of three major nutrients. Citrate, cis-aconitate, isocitrate and Lmalic acid the important intermediates to enter the tricarboxylic acid cycle, in which citrate1 is the product of the rst speed limiting step and plays a role of speed limiting. Citrate1 also plays a role of lens plasma for eyes, bones and sperm (Akram 2014). Isocitrate helps to avoid wheezing and failure of automatic resuscitation under pathological conditions (Rivera-Angulo and Peña-Ortega 2014). L-malic acid is involved in the fourth redox in the tricarboxylic acid cycle, plays a role in promoting the capacity of the tricarboxylic acid cycle (Srere 1975), plays a role in protecting myocardial ischemia/reperfusion injury, anti-in ammatory and anti-latelet aggregation (Tang et al. 2013). Citrate1, cis-aconitate and isocitrate, Lmalic acid decreased signi cantly suggesting that Dnm1 gene deletion may affect the related links in the tricarboxylic acid cycle pathway and further affect energy productivity.
It has been reported that mitochondrial energy production is vital for cell division in addition to other basic functions in the cell, including the regulation of cell volume, cellular architectureand solute concentration (Sweet and Singh 1999). Energy levels differ at various stages of the cell cycle suggesting that there is a relationship between the abundance of energy and the cell's ability to enter a new cell cycle, which supported the hypothesis that mitochondria play a key role in cell cycle regulation (Harbauer et al. 2014; Lopez-Mejia and Fajas 2015). Although the speci c mechanisms between mitochondria and the cell cycle regulation is not well understood, studies have shown that low energy cell cycle checkpoints monitor the energy capability before committing to another round of cell division. There are reports showed that the abnormal mitochondrion division and fusion will make the mitochondrial network become too scattered, which lead to the de ciency of ATP production (Bartolák-Suki et al. 2017). In our experiment, the results of effect of Dnm1 gene deletion on energy in energy metabolism showed that the relative content of Adenosine 5'-triphosphate (ATP) in the Dnm1Δ cells were signi cant lower (P < 0.05) than those in wild type cells. It is suggested that Dnm1 gene deletion had signi cant effect on ATP production of cells.
Our experiment results indicated that the loss of Dnm1 gene from mitochondria resulted in spindle maintenance de ciency, chromosome segregation de ciency, spindle breakage de ciency, coenzyme

Author contributions
This study was designed and conceived by Yiling Hou. The experimental procedures and data analysis were carried out by Xiumei Tan, Xiang Ding and Yiling Hou. The manuscript was prepared by Xiumei Tan, Xiang Ding, Rongmei Yuan and Yiling Hou. All authors read and approved the nal manuscript.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Con icts interest
No competing nancial interests exist.    Notes: Data represented as mean ± SD. ** P<0.01.

Figure 5
The effect of Dnm1 gene deletion on coenzyme in energy metabolism (A-F) Statistical analysis of relative contents of FMN, NAD+, NADP+, TPP, acetyl-CoA and NADPH in wild-type and Dnm1Δ cells (n=3, the experiment was repeated three times). * P<0.05.