Cytoophidia Maintain the Integrity of Drosophila Follicle Epithelium

CTP synthase (CTPS) forms a filamentous structure termed the cytoophidium in all three domains of life. The female reproductive system of Drosophila is an excellent model for studying the physiological function of cytoophidia. Here, we use CTPSH355A, a point mutation that destroys the cytoophidium-forming ability of CTPS, to explore the in vivo function of cytoophidia. In CTPSH355A egg chambers, we observe the ingression and increased heterogeneity of follicle cells. In addition, we find that the cytoophidium-forming ability of CTPS, rather than the protein level, is the cause of the defects observed in CTPSH355A mutants. To sum up, our data indicate that cytoophidia play an important role in maintaining the integrity of follicle epithelium.


Introduction
CTP synthase (CTPS) is a glutamate aminotransferase that catalyzes the transfer of amide nitrogen from glutamine to the C-4 position of UTP. CTP, the product of CTPS, is an important nucleotide and is a component of the synthesis of RNA, DNA, and sialoglycoprotein. It also acts as an energy coupler for some metabolic reactions, such as the synthesis of glycerophospholipids and glycosylated proteins [1,2].
In 2010, CTPS was found to form filamentous structures termed cytoophidia in Drosophila [3]. Subsequently, CTPS has been found to form filamentous structures in bacteria [4] and S. cerevisiae [5]. In the following years, the existence of cytoophidia was confirmed in human cells [6], S. pombe [7], Arabidopsis thaliana [8], and archaea [9], which indicates that cytoophidia are highly conserved in evolution.
Compartmentation is the basis for the function of organelles [10]. The classical cellular compartmentation in eukaryotic cells is achieved through membrane-bound organelles, such as the endoplasmic reticulum, mitochondrion, and Golgi apparatus [11]. Compartmentation establishes a physical boundary for the biological processes within cells, enabling cells to carry out different metabolic activities at the same time, generate specific microenvironments, regulate biological processes in time and space, and determine the specific location where biological processes should occur. The formation of cytoophidia realizes the regionalization of CTPS, and its location in cells may therefore have corresponding physiological significance.
Cells in the Drosophila ovary exhibit vigorous anabolic activity because they need nutrients for development. Cytoophidia are observed from region 2 of the germarium to stage 10A of oogenesis. Based on the widespread presence of cytoophidia in germline cells and follicle epithelial cells of Drosophila ovaries [12], and the characteristics of cytoophidium observed in germline cells and follicle epithelial cells at most stages of oogenesis, Drosophila ovarian follicle cells have become a classic model for studying cytoophidia.
Epidermal tissues form the boundaries of organs, where they perform a range of functions, including secretion, absorption, and protection. These tissues are usually composed of discrete cells, forming a single-cell thick sheet. Follicle epithelium is a simple epithelium. In the process of division, the cells of simple epithelium have a specific orientation of the spindle, so that both daughter cells are located in the epithelial plane. This is considered to be very important for maintaining the integrity of follicle epithelium and preventing hyperplasia [13][14][15][16].
An egg chamber consists of hundreds of follicle cells, and each follicle cell has multiple membrane domains including apical, basal, and lateral. The end adjacent to germline cells is defined as the apical side, and the end far away from germline cells is defined as the basal side [17]. Before mitosis, follicle cells will move toward the apical direction, which may be caused by the extrusion of neighbor cells. This movement results in the displacement of some cells from the tissue layer. Usually, the displaced cells need to be reintegrated to support tissue growth and maintain tissue architecture [18][19][20].
In a previous study, we find that cytoophidia are specifically distributed on the basolateral side of follicle cells, and this specific distribution is related to the polarity regulator of the cell membrane [21]. Therefore, we need to understand the function of cytoophidium which is specifically distributed in follicle cells.
In this study, we describe the effects of cytoophidium disassembly on follicle epithelium integrity. We are also concerned about whether these effects are directly related to the assembly of CTPS into cytoophidia, rather than to the level of CTPS protein. Our results indicate that cytoophidia play an important role in maintaining the integrity of follicle epithelium. In addition, we eliminate the influence of tissue-tissue interaction and find that cytoophidia can directly affect the integrity of follicle epithelium.

CTPS Forms Cytoophidia in Drosophila Follicle Cells
Cells in Drosophila ovaries exhibit vigorous anabolic activity because they need nutrients for development. During Drosophila oogenesis, the follicle epithelium is a sheet of monolayer cells that encase germline cells. CTPS, as the synthase of CTP, plays an important role in the regulation of tissue growth and development. Cytoophidia exist in several different types of cells in the Drosophila ovary from region 2 of the germarium to stage 10A of oogenesis, including epithelial follicle cells ( Figure 1A-C) and germline cells ( Figure 1D-F).

Follicle Cells Undergo Ingression in CTPS H355A Egg Chambers
The amino acid histidine at the 355th position, or His355, lies at the tetramer-tetramer interface of CTPS [22]. If the H355 site is mutated, the cytoophidium cannot be formed. Previous studies showed that the H355 site is essential for its polymerization, but not enzymatic function [23,24]. Our laboratory has solved the structure of Drosophila melanogaster CTPS (dmCTPS) and found that the H355 site lies at the tetramer-tetramer interface and does not affect the catalytic site [25]. Therefore, we constructed an H355A point-mutated knock-in Drosophila strain to investigate whether the disassembly of cytoophidia would affect follicle cells. Former studies found that the H355A served as a dominant negative point mutation [26] ( Figure S1).
In order to find out whether the abnormality is caused by the inability of CTPS to aggregate due to H355A point mutation or the addition of mCherry tag, our laboratory constructed another Drosophila strain with mCherry added to the C-terminus of CTPS based on w 1118 . To determine whether the feature of cytoophidium localization was in fact introduced by protein fusion between CTPS and mCherry tag, we performed immunofluorescence microscopy and directly detected the CTPS protein of the w 1118 fly and found no difference [27]. It is proven by the observation that the knock-in mCherry tag does not affect the polymerization of CTPS protein. The morphology of the CTPS-mCherry KI Drosophila(CTPS-mCh) ovaries is consistent with that of the w 1118 , which implies that the CTPS-mCherry KI Drosophila can also be used as control in our experiment (Figure 2A-F).
Besides, our laboratory has used the CTPS-mCherry KI Drosophila as control in previous studies [28,29].

CTPS Forms Cytoophidia in Drosophila Follicle Cells
Cells in Drosophila ovaries exhibit vigorous anabolic activity because they need nutrients for development. During Drosophila oogenesis, the follicle epithelium is a sheet of monolayer cells that encase germline cells. CTPS, as the synthase of CTP, plays an important role in the regulation of tissue growth and development. Cytoophidia exist in several different types of cells in the Drosophila ovary from region 2 of the germarium to stage 10A of oogenesis, including epithelial follicle cells ( Figure 1A-C) and germline cells ( Figure 1D-F). When constructing the point-mutated Drosophila strain, we added a mCherry tag at the C-terminal of CTPS. Through confocal microscopy, we observed diffuse mCherry signal in Drosophila follicle cells, which confirmed that the CTPS could not form the cytoophidium after mutation at the H355 site ( Figure 3A,F). In the egg chamber of wild-type flies, follicle cells are monolayer epidermal cells. We observed their morphological characteristics by immunofluorescence staining. The cell membrane was labeled with an antibody against Armadillo. We found that in the egg chamber of CTPS H355A/H355A -mCherry knock-in homozygous fly (hereinafter referred to as CTPS H355A strain), some follicle cells originally arranged in a monolayer migrated inward (ie. ingression), thus disrupting the monolayer arrangement. The ingression of follicle cells occurs not only in the early stages of oogenesis, such as stage 5 ( Figure 3A-E), but also in the middle stages of oogenesis, such as stage 8 ( Figure 3F-J). When constructing the point-mutated Drosophila strain, we added a mCherry tag at the C-terminal of CTPS. Through confocal microscopy, we observed diffuse mCherry signal in Drosophila follicle cells, which confirmed that the CTPS could not form the cytoophidium after mutation at the H355 site ( Figure 3A, F). In the egg chamber of wildtype flies, follicle cells are monolayer epidermal cells. We observed their morphological characteristics by immunofluorescence staining. The cell membrane was labeled with an antibody against Armadillo. We found that in the egg chamber of CTPS H355A/H355A -mCherry knock-in homozygous fly (hereinafter referred to as CTPS H355A strain), some follicle cells originally arranged in a monolayer migrated inward (ie. ingression), thus disrupting the monolayer arrangement. The ingression of follicle cells occurs not only in the early stages of oogenesis, such as stage 5 ( Figure    Our study mainly focused on stage 8 egg chambers. We demonstrated the ingression of follicle cells in stage 8 egg chambers through three-dimensional reconstruction ( Figure 4A-C). Combined with the morphological changes of follicle cells observed on the surface of the egg chambers, we speculated that the integrity of follicle epithelia would be disturbed when CTPS could not assemble into cytoophidia. Through statistical analysis, we found that approximately 20% of egg chambers at stage 8 have follicle cells ingression by counting 20 stage 8 egg chambers of each genotype ( Figure 4D). Our results indicate that the Our study mainly focused on stage 8 egg chambers. We demonstrated the ingression of follicle cells in stage 8 egg chambers through three-dimensional reconstruction ( Figure 4A-C). Combined with the morphological changes of follicle cells observed on the surface of the egg chambers, we speculated that the integrity of follicle epithelia would be disturbed when CTPS could not assemble into cytoophidia. Through statistical analysis, we found that approximately 20% of egg chambers at stage 8 have follicle cells ingression by counting 20 stage 8 egg chambers of each genotype ( Figure 4D). Our results indicate that the widely and specifically distributed cytoophidia play a role in maintaining the integrity of follicle epithelia.

Ingressive Follicle Cells Display Abnormal DCAD2 Pattern
In a previous study, we found that cytoophidia are specifically located on the lateral and basal sides of follicle cells [21]. The polarity regulators of follicle cells and adherens junctions have certain effects on the maintenance of cytoophidia. To explore whether the cell membrane components of ingressive follicle cells would be affected when cytoophidium fails to form, we labeled the basolateral regulator Dlg of follicle cells, adherens junctions DE-Cadherin DCAD2, and cell membrane protein Hts. After immunostaining, in the follicle epithelium labeled with Hts and Dlg, there was no significant difference between the cell membrane of ingressive follicle cells and that of normal follicle cells ( Figure 5A-H).

Ingressive Follicle Cells Display Abnormal DCAD2 Pattern
In a previous study, we found that cytoophidia are specifically located on the lateral and basal sides of follicle cells [21]. The polarity regulators of follicle cells and adherens junctions have certain effects on the maintenance of cytoophidia. To explore whether the cell membrane components of ingressive follicle cells would be affected when cytoophidium fails to form, we labeled the basolateral regulator Dlg of follicle cells, adherens junctions DE-Cadherin DCAD2, and cell membrane protein Hts. After immunostaining, in the follicle epithelium labeled with Hts and Dlg, there was no significant difference between the cell membrane of ingressive follicle cells and that of normal follicle cells ( Figure 5A-H).
Under the condition of DCAD2 labeling, we found that DCAD2 showed an abnormal pattern in the ingressive follicle cells compared with normally arranged follicle cells. In normal follicle cells, the end near the germline cells is defined as the apical side and the end near the muscle layer is defined as the basal side. From the cross-sectional view of the lateral side of the stage 8 egg chamber, the DCAD2 pattern should be adjacent to the germline cells. However, in the ingressive follicle cell, DCAD2 could be seen flipping in the direction rather than at the apical side ( Figure 6). and basal sides of follicle cells [21]. The polarity regulators of follicle cells and adherens junctions have certain effects on the maintenance of cytoophidia. To explore whether the cell membrane components of ingressive follicle cells would be affected when cytoophidium fails to form, we labeled the basolateral regulator Dlg of follicle cells, adherens junctions DE-Cadherin DCAD2, and cell membrane protein Hts. After immunostaining, in the follicle epithelium labeled with Hts and Dlg, there was no significant difference between the cell membrane of ingressive follicle cells and that of normal follicle cells ( Figure 5A-H).  Under the condition of DCAD2 labeling, we found that DCAD2 showed an abnormal pattern in the ingressive follicle cells compared with normally arranged follicle cells. In normal follicle cells, the end near the germline cells is defined as the apical side and the end near the muscle layer is defined as the basal side. From the cross-sectional view of the lateral side of the stage 8 egg chamber, the DCAD2 pattern should be adjacent to the germline cells. However, in the ingressive follicle cell, DCAD2 could be seen flipping in the direction rather than at the apical side ( Figure 6).

CTPS H355A Follicle Cells Increase the Heterogeneity
The follicle epithelium of Drosophila consists of a monolayer of follicle cells, which surround the oocyte and 15 nurse cells. Follicle cells gradually differentiate into various subpopulations, which will undergo morphological changes. After stage 6, the follicle cells cease mitosis and are arranged in a hexagonal pattern, which means that under normal circumstances, each follicle cell contacts six adjacent cells, most of which are hexago-

CTPS H355A Follicle Cells Increase the Heterogeneity
The follicle epithelium of Drosophila consists of a monolayer of follicle cells, which surround the oocyte and 15 nurse cells. Follicle cells gradually differentiate into various subpopulations, which will undergo morphological changes. After stage 6, the follicle cells cease mitosis and are arranged in a hexagonal pattern, which means that under normal circumstances, each follicle cell contacts six adjacent cells, most of which are hexagonal and well arranged on the surface of egg chambers. However, in CTPS H355A mutant, the assembly of cytoophidia was disrupted and the number of non-hexagonal cells increased. We segmented the cell by the membrane staining of Hts protein, counting the different shapes cell by cell. In CTPS H355A mutant, we observed many pentagonal follicle cells, and the heptagonal cells increased by about 10% (Figure 7A-D).  The quantification further confirmed that compared with w 1118 , there was difference in the number of heptagonal follicle cells in stage 8 egg chambers of the CTPS H355A mutant. Moreover, the number of hexagonal follicle cells in CTPS H355A egg chambers decreased by about 15% at stage 8, while the number of polygonal cells increased by about 10% (Figure 7E-G). Considering that the more sides the polygon, the closer it is to the round circle, we speculate that when cytoophidia cannot be formed, the cell membrane will be affected, and the tight The quantification further confirmed that compared with w 1118 , there was difference in the number of heptagonal follicle cells in stage 8 egg chambers of the CTPS H355A mutant. Moreover, the number of hexagonal follicle cells in CTPS H355A egg chambers decreased by about 15% at stage 8, while the number of polygonal cells increased by about 10% (Figure 7E-G). Considering that the more sides the polygon, the closer it is to the round circle, we speculate that when cytoophidia cannot be formed, the cell membrane will be affected, and the tight arrangement of epithelial follicle cells will not be maintained.
Morphology changes indicate that cytoophidia located at the basolateral side may play a role in maintaining the integrity of follicle epithelium.

Follicle Epithelia Reduce Compactness in CTPS H355A Mutant
In the wild-type flies, follicle epithelial cells at stages 4-9 of oogenesis are tightly packed, and most of the hexagonal follicle cells enclose germ cells. In the case that CTPS could not be assembled into cytoophidia, we observed that closely arranged epithelial follicle cells became relatively loose, and follicle cells of similar size in the wild-type became relatively very large or very small, which was not conducive to compact arrangement ( Figure 8A-L). In order to further clarify the observed phenomenon, we segmented the follicle cell surface based on the cell membrane and calculated the basal area of each follicle cell on an egg chamber through software. The average area ratio of the group with the largest area of three adjacent follicle cells and the group with the smallest area of three adjacent follicle cells was used as an indicator of follicle cell heterogeneity. The higher the ratio, the higher the heterogeneity of surface follicle cell. The quantitative analysis showed that the average area of follicle cells at stage 8 CTPS H355A was smaller than that of the wild-type, but the heterogeneity was much higher than that of the wild-type ( Figure 8M,N).

Follicle Cell Ingression Occurs in Egg Chamber Overexpessing CTPS H355A
In previous studies, our laboratory found that the formation of cytoophidia can prolong the half-life of CTPS protein in mammalian cells. Therefore, we want to know whether H355A point mutation affects CTPS protein level in Drosophila ovaries. Western blot results confirmed that the level of CTPS protein in Drosophila ovaries after CTPS H355A mutation was lower than that in the wild-type ( Figure 9H,I). Thus, we want to investigate whether the phenotypes observed in the CTPS H355A strain are caused by the decrease of CTPS protein level.
We found that the distribution of cytoophidia in the basolateral side of follicle cells could be clearly observed in Actin > UAS CTPS-mCherry-OE heterozygous egg chambers ( Figure 9A,B). Almost every follicle cell had one or two cytoophidia, and follicle cells were arranged in a single layer. In Actin > UAS CTPS H355A -mCherry-OE homozygous flies, the diffused distribution of CTPS H355A could be observed, and the ingressive follicle cells appeared as well ( Figure 9C-G). These results indicate that loss of the cytoophidiumforming ability of CTPS, rather than its protein level, is the primary cause of follicle cell ingression in the CTPS H355A mutant.
In previous studies, our laboratory found that the formation of cytoophidia can prolo the half-life of CTPS protein in mammalian cells. Therefore, we want to know whether H35 point mutation affects CTPS protein level in Drosophila ovaries. Western blot results confirm that the level of CTPS protein in Drosophila ovaries after CTPS H355A mutation was lower th that in the wild-type ( Figure 9H,I). Thus, we want to investigate whether the phenotypes o served in the CTPS H355A strain are caused by the decrease of CTPS protein level.

Overexpession of CTPS H355A Increases the Heterogeneity of Follicle Cells
Similarly, we wanted to examine whether the heterogeneity of follicle cells was affected by the level of CTPS protein. According to our study, there was a long and curly cytoophidium in each follicle cell on the surface of Actin > UAS CTPS-mCherry-OE heterozygous egg chamber. Compared with the wild-type egg chambers, where cytoophidia are mostly rod-shaped and distributed along the cell membrane, the elongated cytoophidia were still distributed along the cell membrane after the overexpression of CTPS ( Figure 10A-C).

Overexpession of CTPS Increases the Heterogeneity of Follicle Cells
Similarly, we wanted to examine whether the heterogeneity of follicle cells was affected by the level of CTPS protein. According to our study, there was a long and curly cytoophidium in each follicle cell on the surface of Actin > UAS CTPS-mCherry-OE heterozygous egg chamber. Compared with the wild-type egg chambers, where cytoophidia are mostly rod-shaped and distributed along the cell membrane, the elongated cytoophidia were still distributed along the cell membrane after the overexpression of CTPS ( Figure 10A-C). The diffused distribution of CTPS was confirmed on the surface of the egg chamber of Actin > UAS CTPS H355A -mCherry-OE homozygous fly. The changes of morphology and cell size showed that the heterogeneity of follicle cells was enhanced because there was no cytoophidium on the cell membrane. It seemed that these follicle cells could not even be arranged tightly ( Figure 10D-F).

Follicle Cell-Specific Overexpression of CTPS H355A Impairs the Integrity of Follicle Epithelium
After excluding the influence of CTPS protein level, we wanted to further eliminate the effect of the inter-tissue interaction caused by the ubiquitous expression of Actin-Gal4. To this end, we constructed a strain overexpressing CTPS H355A using the same UAS CTPS H355A - The diffused distribution of CTPS was confirmed on the surface of the egg chamber of Actin > UAS CTPS H355A -mCherry-OE homozygous fly. The changes of morphology and cell size showed that the heterogeneity of follicle cells was enhanced because there was no cytoophidium on the cell membrane. It seemed that these follicle cells could not even be arranged tightly ( Figure 10D-F).

Follicle Cell-Specific Overexpression of CTPS H355A Impairs the Integrity of Follicle Epithelium
After excluding the influence of CTPS protein level, we wanted to further eliminate the effect of the inter-tissue interaction caused by the ubiquitous expression of Actin-Gal4. To this end, we constructed a strain overexpressing CTPS H355A using the same UAS CTPS H355A -mCherry-OE and UAS CTPS-mCherry-OE strains together with Tj-Gal4 specifically expressed in follicle cells. As a control, wild-type CTPS was overexpressed specifically in follicle cells using the Tj-Gal4 driver. Our results confirmed that the integrity of follicle epithelium was impaired when CTPS H355A was overexpressed specifically in follicle cells ( Figure 11A-N).

Space between Muscle Sheath and Egg Chamber Increases in CTPS H355A
IF is a member of the integrin complex and widely exists in the muscle layer that encloses the ovarioles. We found that in the wild-type ovary, the muscle sheath tightly wrapped the ovarioles and drove their movement ( Figure 12A,B), which was conducive to common life activities such as oogenesis. In CTPS H355A ovaries, the space between the muscle sheath and the egg chamber was significantly increased, and the egg chamber almost collapsed from the muscle sheath ( Figure 12C-E), which might affect normal physiological activities. mCherry-OE and UAS CTPS-mCherry-OE strains together with Tj-Gal4 specifically expressed in follicle cells. As a control, wild-type CTPS was overexpressed specifically in follicle cells using the Tj-Gal4 driver. Our results confirmed that the integrity of follicle epithelium was impaired when CTPS H355A was overexpressed specifically in follicle cells ( Figure 11A-N).

Space between Muscle Sheath and Egg Chamber Increases in CTPS H355A
IF is a member of the integrin complex and widely exists in the muscle layer that encloses the ovarioles. We found that in the wild-type ovary, the muscle sheath tightly wrapped the

Discussion
To explore the physiological function of cytoophidia in Drosophila follicle cells, we analyze the changes in follicle cells in CTPS mutant when cytoophidia cannot be formed. Our results indicate that the integrity of follicle epithelium is compromised when CTPS lose its cytoophidium-forming capability.
In this study, we generate transgenic flies with a point mutation in CTPS. Mutations do not affect enzymatic activity but lead to the disassembly of cytoophidia. In the mutant flies, the integrity of follicle epithelia is impaired with two related phenotypes: (1) ingression of follicle cells and (2) heterogeneous follicle cells.
We have previously discovered that cytoophidia are specifically distributed on the basolateral side of follicle cells [21]. Moreover, when the polarity of follicle cells is disrupted, cytoophidia will become unstable, especially due to the disruption of apical regulators. In this study, the apical polarity of follicle cells is indeed affected by the absence of cytoophidia. Our data indicate that the cytoophidium, as a kind of membraneless organelle, maintains its specific subcellular localization in biological processes.

Discussion
To explore the physiological function of cytoophidia in Drosophila follicle cells, we analyze the changes in follicle cells in CTPS mutant when cytoophidia cannot be formed. Our results indicate that the integrity of follicle epithelium is compromised when CTPS lose its cytoophidium-forming capability.
In this study, we generate transgenic flies with a point mutation in CTPS. Mutations do not affect enzymatic activity but lead to the disassembly of cytoophidia. In the mutant flies, the integrity of follicle epithelia is impaired with two related phenotypes: (1) ingression of follicle cells and (2) heterogeneous follicle cells.
We have previously discovered that cytoophidia are specifically distributed on the basolateral side of follicle cells [21]. Moreover, when the polarity of follicle cells is disrupted, cytoophidia will become unstable, especially due to the disruption of apical regulators. In this study, the apical polarity of follicle cells is indeed affected by the absence of cytoophidia.
Our data indicate that the cytoophidium, as a kind of membraneless organelle, maintains its specific subcellular localization in biological processes.
In these experiments, we also notice that cytoophidia play a role in maintaining the integrity of follicle epithelium. We speculate that cytoophidia located at the basolateral side of follicle cells may play a role in supporting follicle cells. In the absence of cytoophidia, the mechanical tension of the follicle cell membrane will be reduced, making it more difficult to maintain the cell morphology. Therefore, follicle cells are more likely to be drawn into polygons and expanded by surrounding cells or squeezed and reduced by surrounding cells. Similarly, due to the weakening of membrane mechanical tension, the follicle cells migrating inward after mitosis cannot be reintegrated into the follicular monolayer, resulting in the ingression. Our laboratory also found that in the male reproductive system of Drosophila, when CTPS cannot form cytoophidia, the main cells on the surface of the accessory gland may be difficult to maintain their cell shape, and two horizontally arranged nuclei appear to be vertically arranged. This further support our hypothesis [30].
When cytoophidia are disassembled, the observed separation of egg chamber and muscle sheath may also be due to the disappearance of the supporting force of cytoophidia. When cytoophidia cannot be formed, the internal supporting force of each follicle cell is weakened, leading to the collapse of the entire egg chamber. Considering that the follicle epithelium will develop into the eggshell of a fertilized egg in the later stages [31], it is possible that its shell hardness and the hatchability of the fertilized egg will also be affected accordingly.
However, we could not simply rescue the phenotypes found in the CTPS H355A -mCh mutant by expressing CTPS-mCherry protein. Our previous studies in mammals [26] and Drosophila [27] confirmed that the CTPS H355A point mutation is dominant-negative, which is to say that as long as the CTPS H355A protein exists, the CTPS protein would not be able to assemble into cytoophidium [26,27]. Because the H355A point mutation of CTPS would disrupt the assembly of the cytoophidia dominant negatively, we have analyzed the CTPS H355A/TM6B -mCh egg chambers and found that CTPS H355A/TM6B also have defects in follicle epithelial integrity mentioned above (Supplementary Figure S1). These results further validate our hypothesis that the cytoophidium structure plays a certain role in maintaining epithelial integrity, and the dominant negative CTPS point mutation confirmed that it is crucial for the assembly of cytoophidium.
Since the first discovery of cytoophidia in our laboratory in 2010, great progress has been made in the research on the existence of cytoophidia in different species and different types of cells. However, knowledge concerning the function of this new type of organelle widely existing in organisms is still in the initial stage. Therefore, our work has potential reference value for understanding the role of cytoophidia in Drosophila follicle cells. Our results indicate that forming cytoophidia is crucial to epithelial integrity.

Generation of Transgenic Flies
For polymerase chain reaction (PCR), PUASTattb plasmids were used as the template, and phanta Maxa Super-Fidelity DNA Polymerase (Vazyme, #P505) as the polymerase. Sequences for primers were as below: H355A-F: GAGCAAGTACGCCAAGGAGTGGCAGAAGCTATGCGATAGCCAT; H355A-R: TGCCACTCCTTGGCGTACTTGCTCGGCTCAGAATGCAAAGTTT After obtaining the required plasmids, the CTPS H355A Drosophila strain was constructed by microinjection.

Immunohistochemistry
Ovaries from flies were dissected in Grace's Insect Medium (Gibco) and then fixed in 4% formaldehyde (Sigma) diluted in PBS for 10 min before immunofluorescence staining. The samples were then washed twice using PST (0.5% horse serum + 0.3% Triton × 100 in PBS). For membrane staining, samples were incubated with primary antibodies at room temperature overnight, and then washed using PST. Secondary antibodies were used to incubate the samples at room temperature for another night.

Microscopy and Image Analysis
All images were obtained under laser-scanning confocal microscopy (Zeiss 880). Image processing was performed using Zeiss Zen. ImageJ was used to analyze the area and number of follicle cells.
We used the ImageJ SCF to segment the follicle cells by the membrane, then use ImageJ cell counter to calculate different shapes of cells to get the number of polygonal cells. We used ImageJ to measure the area of each cell. For each statistical quantification, we collected the surface images using Zeiss 880 with the interval as 0.5 µm for z-stack, 5 stage 8 egg chambers were quantified per genotype, biological repeats = 3. Mann-Whitney U test was conducted for comparison.

Western Blotting
Female adult ovaries of Drosophila were collected with gathered into lysis buffer RIPA (Meilunbio, Dalian, China) with protease inhibitor cocktail (Bimake, Shanghai, China) for Western blotting, and then ground with 1 mm Zirconia beads in Sonicator (Shanghai Jing Xin, Shanghai, China). The sample would then lysis on ice for up to 30 min. Samples were centrifuged for 10 min at 10,000 g at 4 • C. The 6× protein loading buffer was pipetted into the supernatants and boiled at 99 • C for 15 min to obtain protein. Then, the protein sample was run through 10% SDS-PAGE gels and transferred to PVDF membranes. At room temperature, membrane was incubated with 5% w/v nonfat dry milk dissolved by 1× TBST for 1 h of blocking. Then, the membrane was incubated with primary antibodies in 5% w/v nonfat milk at 4 • C and gently shaken overnight.
The following primary antibodies were used in this study: anti-mCherry Tag Monoclonal antibodies (Cat. No. A02080, Abbkine, Beijing China), mouse anti-a-Tubulin antibodies (Cat. No. T6199, Sigma). The membranes were washed three times for 5 min per time with shaking, then incubated with secondary antibodies (anti-mouse IgG, HRPlinked antibody, Cell Signaling, Danvers, MA, USA) diluted in 5% w/v nonfat milk at room temperature for 1 h. An Amersham Imager 600 (General Electric, Boston, MA, USA) and Pierce ECL Reagent Kit (Cat. No. 32106, Thermo Fisher, Waltham, MA, USA) were adopted for the chemiluminescence immunoassay. Protein levels were quantified on ImageJ (National Institutes of Health, Bethesda, MD, USA) and normalized to tubulin. At least three biological replicates were quantified.

Data Analysis
Images collected by confocal microscopy were processed using Adobe Illustrator and ImageJ. Cell segmentation based on the cell membrane was achieved using CellPose and SCF methods. Quantitative analysis was processed by Excel and GraphPad. The Mann-Whitney U test was conducted to get the p-value.