The DYRKP1 kinase regulates cell wall degradation in Chlamydomonas by inducing matrix metalloproteinase expression

Abstract

The cell wall of plants and algae is an important cell structure that protects cells from changes in the external physical and chemical environment. This extracellular matrix, composed of polysaccharides and glycoproteins, must be constantly remodeled throughout the life cycle. However, compared to matrix polysaccharides, little is known about the mechanisms regulating the formation and degradation of matrix glycoproteins. We report here that a plant kinase belonging to the DUAL-SPECIFICITY TYROSINE PHOSPHORYLATION-REGULATED KINASE (DYRK) family present in all eukaryotes regulates cell wall degradation after mitosis of Chlamydomonas reinhardtii by inducing the expression of matrix metalloproteinases (MMPs). Without the plant DYRK kinase (DYRKP1), daughter cells cannot disassemble parental cell walls and remain trapped inside for more than 10 days. On the other hand, the DYRKP1 complementation line shows normal degradation of the parental cell wall. Transcriptomic and proteomic analyses indicate a marked down-regulation of MMP gene expression and accumulation, respectively, in the dyrkp1 mutants. The mutants deficient in MMPs retain palmelloid structures for a longer time than the background strain, like dyrkp1 mutants. Our findings show that DYRKP1, by ensuring timely MMP expression, enables the successful execution of the cell cycle. Altogether, this study provides insight into the life cycle regulation in plants and algae.

Keywords: Cell division; Cell wall; Extracellular matrix; Matrix metalloproteinases; Palmelloid; Plant dual-specificity tyrosine phosphorylation-regulated kinase (DYRKP1).

Figure 1.

The Chlamydomonas reinhardtii dyrkp1-1 mutant formed palmelloid structure. A) Cell culture images. B) Distribution of particle diameter (size) in the culture 3 days after inoculation. The mean value of particle diameter was calculated from all particles. C) Confocal microscopic images. The merged images indicate the cell wall stained with Concanavalin A conjugating fluorescent dye at 594 nm (ConcA; cyan) and the chloroplast with chlorophyll autofluorescence at 488 nm (Chl; red). The images clearly showed that the dyrkp1-1 mutant cells were trapped inside 3 parental cell walls surrounded by another big cell wall (white arrows). The yellow scale bar indicates 10 µm. D) Cell population distributions. The composition of particles classified into a single cell and the number of cells inside the parental cell wall. The sample was measured in the culture 3 days after inoculation.

Figure 2.

The dyrkp1-1 mutant showed an impairment in the digestion of the parental cell wall. A) Changes in the total particle volume under standard light condition (80 ± 5 μmol photons m⁻² s⁻¹). Data are presented as means ± SD, n = 6 independent experiments. B) Morphology of pellets after low-speed (500 g) centrifugation. The same volume (10 mL) was collected from the culture of 5 days and 25 days after inoculation. C) Confocal microscopy images of the upper phase and pellet after low-speed centrifugation. The 25-day-old cultures were harvested and the samples were stained with the solution of concanavalin A conjugating fluorescent dye (ConcA; cyan) without filtration. Cells and empty parental cell walls can be distinguished by the presence of chlorophyll autofluorescence (Chl; red). The ConA and Chl signals were obtained at 594 and 488 nm, respectively. The yellow scale bar indicates 20 µm. D) Distribution of particle diameter in the 25-day-old cultures. The population was compared between the autolysin treatment (+Autolysin) and no treatment (Control) groups after 30 min of treatment. The black arrow points to the population of undigested parental cell wall debris. E) The amount of extracellular proteins in the culture medium. Data are presented as means ± SD, n = 3 independent experiments.

Figure 3.

The complemented lines of dyrkp1-1 mutant (DYRKP1-c1 and DYRKP1-c2) appeared the restoration of parental cell wall digestion ability. A) Immunoblot of 137AH strain, dyrkp1-1 mutant, and 2 complemented lines. The upper and below panels indicated DYRKP1 antibody (α-DYRKP1) and loading control, respectively. B) Morphology of pellets after low-speed (500 g) centrifugation. The same volume (10 mL) was collected from the 25-day-old culture. C) Changes in mean particle diameter (size) under standard light condition. Data are presented as means ± SD, n = 6 independent experiments. D) Changes in particle concentration under standard light condition. Data are presented as means ± SD, n = 6 independent experiments.

Figure 4.

The dyrkp1 mutants generated from other cell-walled background strains formed palmelloid structures. A) Cell wall integrity was tested by detergent (Triton X-100) treatment in cell-walled (CW) and cell-wall-less (CWL) background strains. Data are presented as means ± SD, n = 3 independent experiments. B) Immunoblot to DYRKP1 antibody (α-DYRKP1) in the upper panel and loading control in the below panel. C) Distribution of particle diameter in the culture 3 days after inoculation.

Figure 5.

Cell wall proteins and ECM proteases were less abundant in the dyrkp1-1 mutant. A) Schematic diagram of the proteomic analysis workflow. The detailed analysis procedure is shown in Supplementary Fig. S7. B) Hierarchical clustering based on Euclidean distance. Five biological replicates in each group have high similarity to each other. C) The cell wall proteins detected in the upper phase. The cell wall proteins in the upper phase were assumed to be those released from the parental cell walls. Venn diagram represents the number of commonly or uniquely found proteins between the 137AH strain and the dyrkp1-1 mutant. The heatmap shows the protein abundance of major cell wall proteins. Details are shown in Supplementary Fig. S8. D) Heatmap of the matrix metalloproteinases and serine proteases detected in the pellet and upper phase. Statistical analysis was performed using the Student's t-test with 0.05 probability level with Benjamini-Hochberg FDR correction; * P < 0.05, ** P < 0.005 (± SD). “nd”: not detected.

Figure 6.

MMP expression showed a positive correlation to DYRKP1 expression. A) The differentially expressed genes encoding the matrix metalloproteinases and serine proteases. B) Gene expression levels of selected matrix metalloproteinases and serine proteases in 137AH strain and dyrkp1-1 mutant. Data are presented as means ± SD, n = 3 independent experiments. Statistical analysis was performed using the 2-tailed Student's t-test; * P < 0.05. “ns’: no significance. C) Gene expression pattern of DYRKP1, MMP1, MMP3, and MMP13 in 137AH strain, dyrkp1-1 mutant, and 2 complemented lines (DYRKP1-c1 and DYRKP1-c2) under light/dark conditions. Data are presented as means ± SD, n = 3 independent experiments. The value was normalized by the reference gene (RACK1). D) The genomic DNA PCR of MMP knockout mutants from the CLiP library. The left panel indicates the gene structures of MMP1, MMP3, and MMP13. The right panel indicates the PCR band patterns in the background strain (CC5325) and mmp mutants. Red triangles display the primer binding sites to each gene. The details in primers are in the Supplementary Table S2. A bigger size band in mmp mutants than the CC5325 strain indicates the insertion of the paromomycin-resistance gene. E) Distribution of particle diameter (size) in the culture 2 days after inoculation. F) Confocal microscopic images. The merged images indicate the cell wall stained with Concanavalin A conjugating fluorescent dye at 594 nm (ConcA; cyan) and the chloroplast with chlorophyll autofluorescence at 488 nm (Chl; red). The yellow scale bar indicates 10 µm.

Figure 7.

Schematic diagram of the role of DYRKP1 in cell hatching. DYRKP1 induces transcription of MMP1, MMP3, and MMP13 genes located in nuclear DNA. Afterward, the translated protein in the pro-activated form of MMPs (pro-MMPs, circles colored by dark blue and light blue pieces) is delivered through cilia (Long et al. 2016; Zou and Bozhkov 2021) and secreted via an ectosome from the cilia membrane (Wood et al. 2013; Long et al. 2016). Pro-MMPs are activated by other proteases (Wilkinson et al. 2017); for example, pro-MMP1 is activated by VLE1 (circle colored by sky blue) (Kubo et al. 2009; de Carpentier et al. 2022). Activated MMPs then hydrolyze and degrade the hydroxyproline-rich glycoprotein structure of the parental cell wall (Goodenough and Lee 2023), causing fragmentation of the parental cell wall. When the degradation of the parental cell wall progresses sufficiently, the daughter cells are released (cell hatching), and the remaining parental cell wall is completely degraded by MMPs that remain active. The black, green, and purple lines indicate the cell wall, plasma membrane, and nucleus membrane, respectively.