(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Delivery of ceramide phosphoethanolamine lipids to the cleavage furrow through the endocytic pathway is essential for male meiotic cytokinesis [1]

['Govind Kunduri', 'Cancer', 'Developmental Biology Laboratory', 'National Cancer Institute', 'Frederick', 'Maryland', 'United States Of America', 'Si-Hung Le', 'Division Of Metabolomics', 'Medical Institute Of Bioregulation']

Date: 2022-10

Cell division, wherein 1 cell divides into 2 daughter cells, is fundamental to all living organisms. Cytokinesis, the final step in cell division, begins with the formation of an actomyosin contractile ring, positioned midway between the segregated chromosomes. Constriction of the ring with concomitant membrane deposition in a specified spatiotemporal manner generates a cleavage furrow that physically separates the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to intercellular bridges (also called midbody) connecting the 2 dividing cells; however, their biological roles and delivery mechanisms remain largely unknown. In this study, we show that ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in Drosophila spermatocytes and is essential for meiotic cytokinesis. The head group of CPE is also important for spermatogenesis. We find that aberrant central spindle and contractile ring behavior but not mislocalization of phosphatidylinositol phosphates (PIPs) at the plasma membrane is responsible for the male meiotic cytokinesis defect in CPE-deficient animals. Further, we demonstrate the enrichment of CPE in multivesicular bodies marked by Rab7, which in turn localize to cleavage furrow. Volume electron microscopy analysis using correlative light and focused ion beam scanning electron microscopy shows that CPE-enriched Rab7 positive endosomes are juxtaposed on contractile ring material. Correlative light and transmission electron microscopy reveal Rab7 positive endosomes as a multivesicular body-like organelle that releases its intraluminal vesicles in the vicinity of ingressing furrows. Genetic ablation of Rab7 or Rab35 or expression of dominant negative Rab11 results in significant meiotic cytokinesis defects. Further, we show that Rab11 function is required for localization of CPE positive endosomes to the cleavage furrow. Our results imply that endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis.

Funding: This study was funded by the intramural division of the National Cancer Institute, National Institutes of Health (Division of Health and Human Services) to J.K.A. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The study was partly funded with Frederal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024 (to K.N.). The work was partially supported by the Grant-in-Aid for Scientific Research on Innovative Areas (17H06304) [T.B.] and a Grant-in-Aid for Scientific Research (B) (18H01800) [T.B.] from Japan Society for the Promotion of Science (JSPS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

(A) De novo sphingolipid biosynthetic pathway. De novo sphingolipid biosynthetic pathway begins with condensation of an amino acid serine and a fatty acid, lauryl-CoA into 3KDS by SPT complex in the membranes of ER. 3KDS is reduced to dihydrosphingosine by 3KDS reductase. Dihydrosphingosine is acylated to produce dihydroceramide by Schlank. Dihydroceramide is desaturated by Δ4 desaturase (DES1) resulting in the formation of ceramide. Ceramide acts as a precursor for the biosynthesis of complex sphingolipids both in the ER and Golgi complex. Ceramide is actively transported to Golgi by CERT. In the trans Golgi lumen, ceramide is converted to hexosylceramides by GC synthases and to CPE by CPE synthase. (B-D) Sphingolipids were extracted from wild type male and female heads and dissected ovaries and testes using Methanol:Chloroform method and subjected to SFC/MS/MS. WTHM, WTHF, WTOY, and WTTS. Sphingolipids were quantitated by measuring large fragments in MRM, and average amounts from 3 independent biological replicates are shown as picomoles (pmol) in 100 micrograms of carbon. Each sphingolipid species (CPE (1B), Ceramide (1C) and HexCer (1D)) was further subdivided into 5 major subspecies. Each of the subspecies differed in acyl chain length but similar in chemical properties: DHSPH base linked to SFA (DHSPH_SFA), SPH base linked to SFA (SPH_SFA), SPH base linked to MUFA (SPH_MUFA), SPH base linked to PUFA (2–6 double bonds) (SPH_PUFA), and SPD with SFA (SPD_SFA). (E) Cartoon showing the chemical structure of 3 major CPE subspecies including CPE_SPH_SFA, CPE_SPH_MUFA, and CPE_SPD_SFA. (F) CPE subspecies within CPE_SPH_SFA that differed in number of carbon atoms in acyl chains of sphingosine base and fatty acid. (G) CPE subspecies within CPE_SPH_MUFA. (H) CPE subspecies within CPE_SPD_SFA. Statistical significance was calculated using mean, SD, and N in Prism 8. The 2-way ANOVA multiple comparison was used to calculate p-values where **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, and ns p >0.05. Three independent biological replicates were performed for each sample and lipids were extracted from 400 heads or 250 pairs of ovaries or testes for each biological replicate. The data underlying the graphs shown in this figure can be found in S1 Data . 3KDS, 3-ketodihydrosphinganine; CERT, ceramide transfer protein; CPE, ceramide phosphoethanolamine; DHSPH, dihydrosphingosine; ER, endoplasmic reticulum; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SD, standard deviation; SFA, saturated fatty acid; SPD, sphingadiene; SPH, sphingosine; SPT, serine palmitoyltransferase; WTHF, wild-type heads from female; WTHM, wild-type heads from male; WTOY, wild-type ovary; WTTS, wild-type testis.

Ceramide phosphoethanolamine (CPE) is a structural analog of sphingomyelin (SM) in Drosophila melanogaster. CPE is synthesized in the luminal compartment of trans Golgi by ceramide phosphoethanolamine synthase (CPES) ( Fig 1A ). Previously, we have shown that cpes null mutants show significant late pupal lethality during development and only about 25% of mutants survive to adulthood. About 60% of cpes mutant adults have dorsal closure defects and all the mutant males are sterile. Aged cpes mutant adults display light inducible seizures and paralysis due to defective cortex glia [ 27 ]. In this study, we show that CPE has unique acyl chain anchors in spermatocytes and is essential for meiotic cytokinesis, spermatid polarity, and individualization. The head group of CPE is also important for spermatogenesis. CPES expression in transit amplifying to the spermatocyte stage is essential for successful completion of spermatogenesis. We show that aberrant central spindle behavior, but not mislocalization of PIPs at the cleavage furrows, is responsible for the meiotic cytokinesis defect in cpes mutants. Further, we show that endocytically retrieved CPE from the plasma membrane is enriched in Rab7 positive multivesicular bodies that dock to the ingressing membranes and release intraluminal vesicles in their vicinity. Our results demonstrate the importance of CPE-rich membrane addition at the cleavage furrow involving the endocytic pathway.

Cytokinesis is the final step in cell division that divides 1 cell into 2 daughter cells. During cytokinesis, an actomyosin contractile ring is formed that is positioned midway between the segregated chromosomes. Narrowing of this ring with the addition of membrane in a spatiotemporally defined mode produces a cleavage furrow that physically divides the cytoplasm [ 9 ]. During somatic cytokinesis, the 2 daughter cells are interconnected via intercellular bridges prior to abscission. Although these structures are transient, they accumulate distinct lipid species to mediate correct cell division [ 10 – 12 ]. Unlike somatic cytokinesis, in testis, the developing spermatogonial cells divide synchronously with an incomplete cytokinesis where all the daughter cells remain interconnected by cytoplasmic bridges. Thus, the spermatogonial cells develop as a syncytium and become separated from each other only at the end of spermatogenesis during spermatid individualization [ 13 ]. Lipids have been shown to participate in cytokinetic furrow ingression, midbody structure stabilization, and abscission during cytokinesis [ 14 , 15 ]. The high degree of membrane curvature necessary for cleavage furrow ingression and stabilization of intercellular bridges (ICB)/cytoplasmic bridges necessitates lipid components with specific biophysical properties. While the importance of very long chain fatty acids (which are components of sphingolipids and glycosphingolipids) in cytokinesis has been shown, the relationship between membrane lipid composition, ring constriction, and furrow ingression is still underexplored [ 14 , 16 ]. Although sphingomyelins containing very long chain polyunsaturated fatty acids (VLC-PUFAs) have been identified in various mammalian testes including humans, their direct role in spermatogenesis, particularly in meiotic cytokinesis remains unknown [ 17 , 18 ]. Other lipids including PIPs, cholesterol, and phosphatidylethanolamine (PE) have been shown to be enriched at the cytokinetic furrow [ 14 ]. Midbodies also accumulate specific lipids including sphingolipids, phosphatidylserine (PS), phosphatidic acid (PA), and even unique triacylglycerols [ 12 , 15 ]. However, specific mechanisms involved in the delivery of these lipids to the cytokinetic furrow or midbodies remain poorly understood. Membrane traffic via exocytic and endocytic mechanisms has been shown to be essential for successful completion of cytokinesis [ 19 – 23 ]. However, only few studies have focused on individual lipid trafficking via exocytic pathways [ 24 – 26 ] and whether endocytic pathways play direct roles in delivering specific lipids to the cytokinetic furrow or midbody remain unknown.

The head groups of each lipid class have been widely recognized for their biological function. For example, the inositol head group of phosphatidylinositol phosphates (PIPs) and its modifications play critical roles in cellular signaling [ 3 ]. However, even within each lipid class, there are molecularly distinct species that have identical head group but differ extensively in their acyl side chains, such as number of carbon atoms, number of double bonds, and the nature of the chemical linkage (e.g., ester or ether). An increasing number of studies are beginning to reveal the importance of acyl side chains by demonstrating that they selectively bind to target proteins and elicit distinct biological functions [ 4 , 5 ]. For example, a recent study showed that the acyl side chains of diacylglycerol significantly influence the recruitment of protein kinase C to the plasma membrane [ 6 ]. Similarly, the chain length of ceramide was shown to be critical for selective protein cargo sorting at endoplasmic reticulum (ER) exit sites in yeast [ 7 ]. The COPI machinery protein p24 bound specifically to N-stearoyl sphingomyelin (SM C18) and functioned as a cofactor to regulate COPI-dependent transport in the early secretory pathway. Further, this interaction depended on both the head group and the backbone of the sphingolipid [ 8 ].

Eukaryotic cells display high diversity in lipid species that are chemically and structurally distinct. They are classified into 8 major categories, each of which is further subdivided into classes and subclasses [ 1 ]. Lipids usually comprise of a polar head group that is linked by a structural backbone to a hydrophobic tail composed of acyl chains. Cellular lipid diversity arises from the large number of combinatorial chemical possibilities that the head and tail moieties can generate. Diversification of lipids to cope with evolving cellular complexity has resulted in thousands of lipid species with purported unique functions, most of which still remain unknown [ 2 ].

Results

CPES enzymatic activity is required for spermatogenesis and accumulation of ceramide is not the cause for spermatogenesis defects To determine whether enzymatic activity of CPES is required for spermatogenesis, we generated an active site mutant of CPES. CPES has a conserved amino acid motif D(X) 2 DG(X) 2 (A/Y)R(X) 8-16 G(X) 3 D(X) 3 D in the CDP-alcohol phosphotransferase (CAPT) domain [40]. The final aspartates of this motif were shown to be essential for catalysis of human choline/ethanolamine phosphotransferase CEPT1 [41,42]. We substituted the last 2 active site aspartates of CPES with alanine residues and subcloned into pUAST vector for generating transgenic flies. The UAS_CPES (DX 3 D to AX 3 A) mutant transgene was expressed in cpes mutant background using bam-Gal4 to test if spermatogenesis phenotypes could be rescued. As shown in S2A and S2B Fig, the active site mutant did not rescue spermatogenesis phenotypes suggesting a crucial role for CPES enzymatic activity in spermatogenesis. We next investigated whether accumulation of ceramide at the Golgi is responsible for the observed phenotypes. Ceramide is generated in the membranes of ER via the de novo biosynthetic pathway catalyzed by the rate-limiting enzyme serine palmitoyltransferase (SPT) (Fig 1A). Subsequently, ceramide is actively transported from ER to Golgi via ceramide transfer protein (DCERT) and thus absence of DCERT could prevent ceramide accumulation at the Golgi (Fig 1A) [43]. To investigate whether blocking ceramide transport from ER to Golgi could restore spermatogenesis, we generated cpes and dcert1 double mutants and their testes were analyzed for meiotic cytokinesis and spermatid polarity phenotypes. However, as shown in S2C and S2D Fig, cpes; dcert1 double mutants did not rescue meiotic cytokinesis and spermatid polarity indicating ceramide accumulation at the Golgi (due to lack of CPES activity in the Golgi) may not be responsible for cpes mutant phenotypes. In Drosophila, transgenic expression of neutral ceramidase was shown to reduce ceramide levels in vivo [44]. We overexpressed ceramidase (UAS CDase) in germ cells using bam-Gal4 in cpes mutant background and testes from the resulting progeny were analyzed for the rescue of mutant phenotypes. However, expression of CDase did not rescue meiotic cytokinesis and spermatid polarity (S2E and S2F Fig), suggesting that absence of CPE but not accumulation of ceramide is responsible for the phenotypes.

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001599

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.