VMP1-deficient Chlamydomonas exhibits severely aberrant cell morphology and disrupted cytokinesis
© Tenenboim et al.; licensee BioMed Central Ltd. 2014
Received: 21 January 2014
Accepted: 28 April 2014
Published: 6 May 2014
The versatile Vacuole Membrane Protein 1 (VMP1) has been previously investigated in six species. It has been shown to be essential in macroautophagy, where it takes part in autophagy initiation. In addition, VMP1 has been implicated in organellar biogenesis; endo-, exo- and phagocytosis, and protein secretion; apoptosis; and cell adhesion. These roles underly its proven involvement in pancreatitis, diabetes and cancer in humans.
In this study we analyzed a VMP1 homologue from the green alga Chlamydomonas reinhardtii. CrVMP1 knockdown lines showed severe phenotypes, mainly affecting cell division as well as the morphology of cells and organelles. We also provide several pieces of evidence for its involvement in macroautophagy.
Our study adds a novel role to VMP1's repertoire, namely the regulation of cytokinesis. Though the directness of the observed effects and the mechanisms underlying them remain to be defined, the protein's involvement in macroautophagy in Chlamydomonas, as found by us, suggests that CrVMP1 shares molecular characteristics with its animal and protist counterparts.
KeywordsVMP1 Autophagy Cytokinesis
VMP1 (Vacuole Membrane Protein 1) is a transmembrane protein with homologues in all eukaryotic kingdoms, with fungi being a notable exception. It has been shown to be located in several cellular compartments: the Golgi apparatus , the endoplasmic reticulum [1, 2], autophagosomes , and the plasma membrane . It has been implicated in an array of cellular processes: organellar biogenesis, endo-, exo-, phagocytosis and protein secretion ; apoptosis ; adhesion ; and, perhaps most notably, macroautophagy (henceforth: "autophagy"; ), as well as a special type of autophagy, namely zymophagy, in which zymogen granules—subcellular structures that contain inactive digestive enzyme—are selectively degraded . Last but not least, it has been shown to play a key role in certain human diseases, namely pancreatitis [1, 6], diabetes , and several types of cancer [4, 8, 9]. The phenotypes and processes in which VMP1 is involved are gradually being elucidated at the molecular level: its plasma-membrane localization and cell-adhesion capacity, for instance, account for its reduced expression in metastasized cancer cells , since loss of adhesion is essensial for metastasis ; and its role in zymophagy helps to protect pancreas cells from digesting themselves during pancreatitis .
In this work we identified a VMP1 homologue (henceforth named CrVMP1) in the green alga Chlamydomonas reinhardtii. Partial silencing of the CrVMP1 gene resulted in severe phenotypes, mainly affecting cell division and morphology. VMP1 mutants in other organisms have thus far never shown any detrimental effects concerning these two cellular aspects, raising curious questions regarding the evolution of the protein's role across the various eukaryotic kingdoms.
VMP1 knockdown cells show severe phenotypes
We used artificial miRNA [18, 19] in an attempt to silence CrVMP1. Two C. reinhardtii strains were used for silencing: CC-4350, better known as cw15 302, a cell-wall-deficient strain that shows high transformation efficiencies; and UVM11, a derivative of CC-4350 that had undergone UV mutagenesis with the purpose of enhancing its capacity for expressing foreign genes . We analyzed CrVMP1 mRNA levels in our lines using qRT-PCR. In transformed clones mRNA levels varied greatly; all the clones we used in our experiments exhibited CrVMP1 mRNA levels that ranged between 5–25% of WT and of empty-vector control.
Additionally, and often concomitantly, cells exhibited aberrant organelle numbers. The organelles in question were pyrenoids (Figure 3C,H) and eyespots (Figure 2E,G,H,K; Figure 3B,C,E,G), of which many of our knockdown cells had two or more (compared with one pyrenoid and one eyespot in WT cells), contractile vacuoles (Figures 2C,I; Figure 3C,D,E,F; two or more pairs in knockdowns, one pair in WT), and nuclei (Figure 3L; two in knockdowns, one in WT). The latter were detected by staining the cells with the fluorescent DNA dye 4',6-diamidino-2-phenylindole (DAPI), followed by epifluorescence microscopy (Figure 3L). The binucleated cells could hardly be mistaken for WT cells undergoing normal mitosis: the nuclei were abnormally positioned, the cells were perfectly round and devoid of cleavage furrows, and lastly, the images were taken at a time point in which almost no cell underwent mitosis, indicating that the binucleated cells were a several-hour-old remnant from the previous round of division.
Multiple pyrenoids per cell were frequently easily discernible in brightfield microscopy. In epifluorescence microscopy, however, many cells that looked normal in brightfield and whose pyrenoid number was hard to determine visually would often reveal, by means of the characteristic cavity in the center of the red-autofluorescing chloroplast, that they indeed harbored two pyrenoids (Figure 3M). The aberrant numbers of nuclei, pyrenoids, eyespots and CVs again suggest defective cytokinesis.
Many of our mutant cells displayed aberrant cell shapes and internal structures. Cells of the former category failed to maintain the usual round or oval external shape of WT C. reinhardtii, but rather appeared in an impressive variety of irregular shapes (Figure 2E,F,I,J,K). In cells with aberrant internal features, the usual structures and organelles that are the trademark of WT C. reinhardtii, especially the cup-shaped chloroplast, appeared deformed and misshapen (Figures 2 and 3). We postulate that defective cytokinesis again was the culprit, as organelles misdeveloped and undetached daughter cells pressed against each other to cause deformities. This, however, bears further investigation.
Lastly, two additional phenotypes appeared on occasion: a small proportion of the cells were highly vacuolated, presumably an indication of undergoing cell death (Figure 2B). More rarely, cells were brimming with numerous globules, deeming any organelle unrecognizable (Figure 3K). This latter phenotype appeared also in WT cells, though much more rarely than in the mutant.
All the mentioned phenotypes appeared both in strain CC-4350 and UVM11, with a tendency of the former toward gross deformities and the latter toward milder, WT-like cell forms with double pyrenoids and nuclei. No correlation was observed between residual mRNA levels, as mediated by qRT-PCR, and phenotypic strength. Penetrance never reached 100%; great variation in the ratio of cells showing aberrant phenotypes was observed. We found no correlation between growth conditions (temperature, light intensity and regime, shaking speed, carbon source, culture age) and penetrance. Although CrVMP1 may be potentially involved in the cell cycle and thus be differently expressed in different stages of the cycle, we saw no difference in phenotypic strength or in CrVMP1 mRNA levels when assayed periodically in an attempt to capture representative stages (data not shown). The milder phenotypes, such as double pyrenoids and nuclei, appeared at times in up to 90% of the cells, whereas extreme deformities showed a penetrance of around 20% (Additional file 1: Table S1). We hypothesize that the lower penetrances were a combined result of low visibility of some of the phenotypes, the influence of the cell-cycle stage of individual cells, as well as stochastic effects.
The cytokinetic defects notwithstanding, our mutants were not notably retarded in growth. Mutant liquid-cultures grew slightly, but not significantly, more slowly, and after a while always reached WT cell-densities (data not shown), indicating that most mutant cells eventually underwent successful cytokinesis. In addition, our mutants exhibited no defects in the CV or in osmoregulation, as reported in VMP1-deficient slime mold . To test this, cells were immersed in pure water and incubated for several hours, with WT and mutant showing similar survival rates (data not shown). Incidentally, the clearly visible CVs in our images often helped us orient inside the more grossly deformed cells, in which organelles and cell polarity were hardly recognizable (for instance, Figure 2B,F,I; Figure 3D,E,I).
Electron microscopy reveals internal cellular defects
Several cell-cycle and autophagy regulators are underexpressed in VMP1 mutants
Metabolomic profiling reveals additional changes in mutant
VMP1 has been researched before in a plant (A. thaliana; ) and in a microorganism (D. discoideum; ), and now: in a species that is both. In this work we were able to assign a novel role to this already versatile protein. VMP1 has thus far not been implicated in relation to defective cell division. At the same time, a rather modest number of cytokinesis mutants in Chlamydomonas has been reported . Some were natural isolates , others generated by and identified following nonspecific mutagenesis , while others yet were the result of targeted silencing [28–30]. In the three latter examples, proteins of the basal bodies, cytoskeleton, and flagella, respectively, were silenced. Indeed, these three structures are tightly related to cell division; processes such as flagellation and cleavage-furrow formation, formerly thought to be completely distinct, are gradually losing the borders between them, and proteins that are associated to these structures often exhibit multiple roles. Not much can be deduced though with regard to the cytoskeleton, basal bodies and flagella in our mutant. As for the latter, our background Chlamydomonas strains, CC-4350 and UVM11, harbor very short flagella; often no flagella were observed under the microscope, anecdotally more so in the mutant than in WT, but it was impossible for us to determine whether this should be ascribed to the VMP1 deficiency or rather to limited visibility.
Another organelle starring in our knockdown cells was the eyespot (Figures 2 and 3). It has been reported that a few cells with two eyespots can frequently be seen in WT cultures. True mutants that harbor multiple eyespots (as well as mutants that have unusually small eyespots and ones that have no eyespots at all) have also been reported . Our mutant cells were markedly different: they almost always showed cell-division defects (cleavage furrows, deformed chloroplasts) in addition to harboring multiple eyespots; and they lacked the mlt1 − characteristic of usually having one correctly placed eyespot and an additional, aberrantly placed eyespot near the base of the flagella .
Electron microscopy revealed in the mutant enlarged mitochondria, disorganized chloroplasts, autolysosomes in WT but not in the mutant, overnumbered and perhaps misshapen Golgi apparatuses, and excessively large starch granules (Figures 4 and 5). The latter two phenomena are known to be linked: plants tend to accumulate starch when their Golgi system is inhibited or disrupted . However, claiming that such is the case in our mutant would be unsubstantiated: our EM images do not allow for a clear-cut definition of the Golgi's well-being or lack thereof in our cells. That said, the increased number of Golgi apparatuses in our mutant (Figure 5C,E) points again toward abnormality. The same applies for starch: enzymatic quantification showed no difference in starch content between WT and mutant (Additional file 2: Figure S1), and our EM images allow no statistical analysis of the amount or size of starch granules. This aspect of our phenotype therefore remains anecdotal and bears further investigation. A connection between VMP1 and Golgi in Chlamydomonas would correspond to observations in the former's homologues: VMP1 was shown to be localized to the Golgi apparatus in the first VMP1 study , and VMP1-deficient slime mold showed morphological and functional Golgi defects .
VMP1 has been shown several times to be an inducer of autophagy, and the molecular mechanisms by which it so acts are gradually being elucidated . It has been shown that VMP1, a multispan transmembrane protein, is anchored to the ER membrane and is essential for autophagosome formation, as siRNA-VMP1 knockdown cells hardly form any autophagosomes, even under autophagy-inducing conditions, such as starvation and rapamycin treatment . Autophagy, despite being extensively researched and discussed—albeit mostly in animals—still bears many unknowns; doubly so in Chlamydomonas, in which the topic is in its infancy . In our study we tentatively show that autophagy may be downregulated in Chlamydomonas VMP1 knockdown cells (it should be noted that autophagy was never actively induced in our experiments. The observed autophagic phenomena in our WT cells represent basal autophagy, possibly in combination with slight, unintentional nutrient deprivation as a function of the culture's age). With regard to the observed underexpression of autophagy markers (Figure 6B), we identified with interest VMP1's interaction partner in humans, beclin-1 (named ATG6 in yeast), as one of the genes downregulated in our knockdown. The VMP1-beclin-1 interaction in human was shown to be essential for the induction of autophagy . It would be of interest to test whether a regulatory adaptation of beclin-1 levels to VMP1 levels in the cell accounts for the former's decreased levels in our mutant.
Further evidence for compromised autophagy was delivered by TEM in the form of autolysosomes that were present in WT but nearly absent in the mutant (Figures 4 and 5), and of grossly enlarged mitochondria in the mutant (Figure 5D). It has been shown that inhibited autophagy results in the accumulation of enlarged mitochondria in rat myoblast cells . Mitochondria frequently undergo fission in healthy cells, but a fraction fail to do so, for unknown reasons. These larger, non-fissioned mitochondria are presumably discriminated against by the autophagic (more precisely: mitophagic) machinery, since the engulfment of large organelles by autophagosomes requires more energy than that of smaller organelles. This is exacerbated by compromised autophagy, such as occurs in scenecent cells or in experimental inhibition of autophagy: clearance of larger mitochondria by means of autophagy is reduced even more, and soon enough they become a majority . The increased levels of reactive oxygen species that enlarged, damaged mitochondria produce  in turn damage the autophagic machinery even more.
The accumulation of starch, as seen in our TEM images, may also be a sign of disrupted autophagy: the aberrant accumulation of glycogen, starch's animal counterpart, in three genetic human diseases—Lafora disease , Pompe disease , and Danon disease —with detrimental pathophysiologies, has been shown in the cited studies to be a result of compromised autophagy. More recently and relevantly, disrupted/inhibited autophagy resulted in starch accumulation in leaves of Arabidopsis thaliana and Nicotiana benthamiana. Indeed, no excess of starch was found in our mutant when quantified enzymatically (Additional file 2: Figure S1); however, the accumulation of starch in excessively large granules may not necessarily correspond to an overall increase in cellular starch content. Our last piece of evidence is reflected in the results of our lipidomic profiling (Figure 7B), whereby the accumulation of TAGs in our mutant may be ascribed to defective autophagy , a result, according to the cited study, of reduced hydrolysis of TAGs by defective autophagosomes. The changes we observe in other classes of lipids may also be related to autophagy, especially in the context of lipids that are constituents of the autophagosome and autolysosome membranes. Out of six phosphatidylethanolamine (PE) species that we deteceted, four were reduced in the mutant (two considerably, two mildly), one was unchanged and one accumulated in the mutant. PE is the major lipid involved in the initial stages of autophagosome formation, and it conjugates with ATG8 . However, conclusions to be drawn from that must remain tentative: it is not known how paucity or plethora of autophagosomes influence the overall levels of PE in the cell; it is also not known which other lipid classes are major components of autophagosomes and autolysosomes. Incidentally, PE has also been shown to be essential for cytokinesis: Chinese hamster ovary cells depleted of PE showed severe cytokinesis defects .
Further experiments are required in order to provide a more definite conclusion in regard to the autophagic capacity of our mutant: ATG8 is better used in its Western-blot rather than its qRT-PCR version as a marker for autophagy; additionally, numerous reasons can account for the accumulation of TAGs, indeed a common stress response in many organisms; a direct link between the accumulation that we observe and defective autophagy still requires establishment. In this regard, our results seem to contradict several studies that showed that the massive TAG and starch accumulation that occurs in nitrogen-deprived Chlamydomonas is accompanied by very pronounced autophagy . If TAG and starch accumulation go hand in hand with autophagy, why do we observe the two former phenomena in our supposedly autophagy-compromised cells? We postulate that the effects reported in the cited, and other, studies, indeed prerequisite nitrogen starvation, a dramatic onslaught that forces the cell to degrade proteins in order to replenish its amino-acid stock, this by means of autophagy. Basal autophagy, such as in our experiments, presumably behaves differently. As mentioned above, Singh et al.  found that disrupted autophagy results in TAG accumulation in rat and mouse, and, conversely, that induced autophagy results in decreased TAG levels. This has been shown in Chlamydomonas as well: applying the autophagy inhibitor 3-methyladenine resulted in TAG accumulation in both nitrogen-deprived and control cells , whereupon the author concluded that TAG accumulation in nitrogen-deprived cells may not be a direct result of autophagy.
Another observation combines our microscope images and our lipidomic profiling. A small fraction of our cells displayed a highly globulized phenotype (Figure 3K). This occurred in WT as well, but much more frequently in the VMP1 knockdown. These cells greatly resemble the lipid-body accumulating cells, following nitrogen deprivation, seen in Goodson et al. . Presumably nitrogen deprivation occurred in a fraction of our cultures, resulting in massive accumulation of lipid bodies, which, incidentally, contain mainly TAGs (such cultures, it should be noted, were never used in any other experiment). The fact that this occurred much more frequently in the VMP1 knockdown reinforces the finding of TAG accumulation mediated by our analytical profiling, and again contradicts, if indeed autophagy is compromised in our cells, the findings reported by Wang et al. .
Our results show concurrence with observations in other organisms with regard to VMP1's involvement in autophagy, but also suggest a heretofore unreported involvement in cell division. It is interesting to note that despite the relatively high sequence conservation between the different VMP1 homologues, CrVMP1 occupies a phylogenetic branch distinct from the other six reported homologues (Figure 1C). Plausibly its sequence divergence is responsible for its added roles in Chlamydomonas. That said, the two hallmarks of our mutant phenotype, defective cytokinesis and defective autophagy, may be tightly linked to each other. Two recent reports provide such a link. In the first, inhibition of autophagy resulted in cytokinesis failure, multinucleation and aneuploidy in human cells . According to this report, the cell uses autophagy to maintain correct levels of RhoA, a small GTPase protein critical for cytokinesis. The second report shows that inhibited autophagy in yeast results, under conditions of starvation, in aneuploidy, abnormal mitosis, and growth deficiency . An even more direct link between cytokinesis and VMP1's specific role in autophagy is provided by phosphatidylinositol 3-phosphate (PI3P). The synthesis of this lipid at the preautophagosomal site is essential for autophagosome formation and is mediated by the enzyme PI3-kinase class III, which forms a complex with, among others, VMP1 . PI3K-III has been shown to form complexes that regulate cytokinesis, and that are very similar to the autophagy-related PI3K-III complexes. Downregulation of various components of the PI3K-III complex resulted in impaired cytokinesis [50, 51]. It is therefore possible that cytokinesis in our mutant is inhibited either as a result of impaired autophagy, whereby the VMP1 deficiency is solely the cause for said impairment; or that the VMP1 deficiency causes the cytokinesis defect in a more direct fashion, possibly through the disruption of PI3K-III complexes. Thus far, VMP1's strong involvement in cancer in humans, a disease whose hallmark is cell division gone awry, has been shown to be a result of the protein's roles in cell adhesion  and in autophagy [52, 53]. Our results may point toward a more direct role of VMP1 in cell division.
In this study we generated VMP1-deficient Chlamydomonas and subsequently identified both a novel role for VMP1 and a new gene involved in Chlamydomonas cell division and autophagy. CrVMP1-deficient cells exhibited disrupted cytokinesis and aberrant morphologies. Cell-biological and biochemical evidence suggests impaired autophgay: mutant cells accumulated triacylglycerides and formed enlarged starch granules; they were devoid of autolysosomes although WT cells showed many; and they showed enlarged mitochondria. In addition, mutants underexpressed several key genes involved in cell-cycle regulation and autophagy.
Strains and cultivation
The cell-wall-deficient C. reinhardtii strains CC-4350 (cw15 302) and UVM11  were used in this study. Cells were grown in Tris-acetate-phosphate (TAP) medium  and were cultured in 22°C, with light intensity of 85 μmol/m2/sec, while shaking at 125 rpm, and, unless stated otherwise, under continuous light. Growth plates were prepared with TAP and 1.5% agar.
Generation of VMP1 knockdown lines
Gene silencing was performed by amiRNA as described . Briefly, the following pair of oligonucleotides was generated by submitting the identifier 187038 to the online WMD3 tool  (http://wmd3.weigelworld.org/): CTAGTTTGAGGTAGAGGATCTTTCAATCTCGCTGATCGGCACCATGGGGGTGGTGGTGATCAGCGCTATTGAGGGATCCTCTACCTCAAG and CTAGCTTGAGGTAGAGGATCCCTCAATAGCGCTGATCACCACCACCCCCATGGTGCCGATCAGCGAGATTGAAAGATCCTCTACCTCAAA. The oligonucleotides were annealed to each other, then ligated into the SpeI restriction site of the pChlamiRNA2 and pChlamiRNA3int vectors. The vectors were linearized and transformed into CC-4350 and UVM11 cells, respectively, using glass beads . Transformant selection was done by arginine auxotrophy for CC-4350 and paromomycin for UVM11.
Measurement of gene expression using qRT-PCR
Total RNA was isolated using the Universal RNA Purification Kit (Roboklon, Berlin, Germany). cDNA synthesis was performed with the RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, Schwerte, Germany) using 1 mg total RNA. qRT-PCR reactions were set using the SYBR Green master mix (Applied Bioscience, Darmstadt, Germany) and performed on an HT 7900 cycler (Applied Bioscience, Darmstadt, Germany). Ubiquitin and RACK1 were used, always simultaneously, as reference genes. Relative expression values were calculated using the 2–ΔΔCt method . Primers were designed using QuantPrime . The primers used for the detection of VMP1 mRNA are TGACGGACTGAGTTGGAAAGGC and GAGCTAGAGGCTTCTTGCGTTG. All other primers are listed in Additional file 5: Table S3.
Light and fluorescence microscopy were performed on an Olympus (Hamburg, Germany) BX-51 microscope with an oil-immersion × 100 objective, using Nomarski optics. Cells were prepared for inspection as described . For transmission electron microscopy, Chlamydomonas cells were fixed for 3 h at 4°C with 2% (v/v) glutaraldehyde in 0.1 M sodium cacodilate buffer (pH 7.4). Samples were then fixed and osmicated for 1 h in 1% (w/v) OsO4. They were then stained for 2 h in 2% (w/v) uranyl acetate and dehydrated progressively in 50, 75, 95, and 100% ethanol, followed by two washes in 100% propylene oxide. The samples were embedded in Spurr's low-melting epoxy resin. Samples were degassed and cured at 60°C for 24 h. Sections (50 nm) were obtained with a Leica UC 6 ultramicrotome (Leica Microsystems, Wetzlar, Germany), mounted on 200-mesh nickel grids, counterstained with uranyl acetate followed by lead citrate, and examined with an energy-filter transmission electron microscope (EFTEM) at 80 kV (Zeiss, Oberkochen, Germany).
Enzymatic starch quantification
Starch was quantified as described . Briefly: liquid Chlamydomonas cultures were centrifuged, frozen in liquid nitrogen, resuspended in 80% (v/v) ethanol, then ultrasonicated. Homogenates were heated to 80°C, centrifuged, then lyophilized. Pellets were washed with water, then resuspended in 0.2 N KOH and heated to 95°C for 1 h. 1 N acetic acid was added and the samples were centrifuged. Supernatants were mixed with amyloglucosidase and incubated at 55°C overnight. Glucose was then enzymatically quantified according to Stitt et al. .
Gas chromatography and mass spectrometry (GC-MS) analysis was performed as described . Briefly: six biological replicates from each strain were subjected to GC-MS analysis. Metabolite levels were determined using the TargetSearch software package . The metabolites' retention indexes (+/−2 s) and spectra (similarity > 85%) were compared against compounds stored in the Golm Metabolome Database (GMD ). Secondary metabolites and lipids were extracted and analyzed as described  using Waters Acquity ultra-performance liquid chromatography (UPLC) coupled to a Thermo-Fisher Exactive Fourier-transformation mass spectrometer. Obtained raw chromatograms were processed using Xcalibur (Thermo-Fisher, Bremen, Germany) and Refiner MS (GeneData, Basel, Switzerland). For peak annotation, the locally developed GoBioSpace database was used . Peak intensities were normalized to the total ion count.
We thank Eugenia Maximova, Änne Eckart, Antje Bolze and Gudrun Wolter for excellent technical assistance, and Tamar Avin-Wittenberg, Michael Schroda, André Scheffel and Sabeeha Merchant for valuable scientific discussions. We acknowledge the financial support of the Investitionsbank des Landes Brandenburg (ILB) (project number 80139585).
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