Qassem A et al. (2025), Possible role of microtubules in vesicular tran...

ECB-ART-54188

Dev Dyn 2025 Aug 04; doi: 10.1002/dvdy.70068.

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Possible role of microtubules in vesicular transport of matrix protein during sea urchin larval biomineralization.

Qassem A, Gildor T, de-Leon SB.

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BACKGROUND: Biomineralization is a vital biological process through which organisms produce mineralized structures such as shells, skeletons, and teeth. Microtubules are essential for biomineralization in various eukaryotic species; however, their specific roles in this process remain unclear. RESULTS: Here, we investigated the structure and function of microtubule filaments and their co-localization with matrix and focal adhesion proteins during the elongation of the calcite spicules of the sea urchin larva. First, we show that inhibiting microtubule polymerization using Nocodazole in whole embryos and isolated skeletogenic cell cultures results in a significant reduction of skeletal growth and affects skeletal morphology. Next, we demonstrate that microtubule filaments elongate from around the skeletogenic nuclei to the biomineralization compartment where they overlap with active focal adhesion kinase. The expression of spicule matrix proteins overlaps with microtubule filaments around the nuclei and with microtubule filaments that elongate to the spicule cavity. CONCLUSIONS: We propose that vesicles bearing matrix proteins are trafficked on microtubules to the spicule cavity where their exocytosis is assisted by focal adhesions. The role of microtubules in biomineralization from unicellular algae to human bones suggests that the proposed microtubule-guided vesicle transport into the biomineralization compartment could be a common mechanism in Eukaryotes' biomineralization.

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	FIGURE 1. Perturbation of microtubule elongation interferes with skeletal growth. (A–E) Nocodazole treatment added at 25hpf in whole embryos, scored at 48hpf. (A) Representative control sea urchin embryo showing normal body rod, PO – post oral rod and AL – anterolateral rod. (B–D) Representative embryos treated with Nocodazole. (B) Embryo showing delay in skeletogenesis, (C) severely delayed embryo where skeletal grains are formed, and (D) malformed embryo that lacks skeleton. (E) Percentage of embryos showing the phenotypes for different Nocodazole concentrations. The number of biological replicates is n = 8 for control; n = 5 for 166, 100, and 33 nM Nocodazole, and n = 3 for 16.6, 6.6, and 0.33 nM Nocodazole. In each biological replicate, 21–66 embryos were scored. Color code matches the phenotypes presented in (A)–(D). Error bars indicate standard deviation. (F–K) Nocodazole treatment added at 48hpf in skeletogenic cell cultures scored at 72hpf. (F) Representative control linear spicule, (G) representative branched spicule under 166 nM Nocodazole treatment. (H) Representative curved spicule under 33 nM Nocodazole, (I) representative branched and curved spicule under 33 nM Nocodazole. (J) Percentage of embryos showing the phenotypes for control and different Nocodazole concentrations. (K) Measurement of spicule length in skeletogenic cell culture at 72hpf. Each box plot shows the average marked in x, the median, the first and the third quartiles (edges of boxes), and all experimental measurements (dots). Number of spicules measured n = 161 in control, n = 84 in 33 nM, n = 113 in 166 nM, and n = 189 in 330 nM Nocodazole treatment. Statistical significance was measured using a two‐tailed t‐test and * indicated p < .05, and ** p < .01.
	FIGURE 2. Microtubules organization in the skeletogenic lineage during spicule formation, triradiate growth and elongation. Representative images showing sea urchin embryo stained with β‐Tubulin (green) and the skeletogenic cell marker 6a9 (blue) at 24hpf (A–H), 27hpf (I–P), and 33hpf (Q–W). (A) and (E) show different focal plains of the same embryo at 24hpf. (B–D) Zoom‐in to the spicule area marked with white rectangle in (A). (F–H) Zoom‐in to the pseudopodia cable area, marked with white rectangle in (E). (I, M) Different focal plains of the same embryo at 27hpf. (J–L) Zoom‐in to the mineralized spicule area rectangle marked in I. (N–P) zoom‐in into the pseudopodia cable area marked in (M). Arrows point to skeletogenic cells where the microtubules elongate from a crescent shape around the nucleus to the cell cortex. Arrowheads point to the spicule cavity (B–D, J–L) or to the empty pseudopodia cable (F–H, N–P). (R–W) Enlargements of the areas marked with white rectangles in Q, showing the body rod region (R–T) and post‐oral rod region (U–W). Arrows point to skeletogenic cells where the microtubules are enriched in a crescent shape around the nuclei and multiple filaments elongate toward the tips of the spicule rods. Arrowheads point to skeletogenic cells away from the tips, not showing the elongated microtubule filaments. Number biological replicates and total embryos scored are, n = 3, 17 at 24hpf, n = 3, 20 at 27hpf, n = 4, 38 at 33hpf. Scale bar in (A, D, E, H, I, L, M, P, T, W) is 10 and 50 μm in (Q).
	FIGURE 3. Microtubule filaments elongate toward the tip of the rods, at 48hpf. (A–C) Representative images showing sea urchin embryo stained with Tubulin‐β (green) and the skeletogenic cell marker 6a9 (blue) at 48hpf. (D–L) Enlargement of the areas marked with white rectangles in C, showing the body rod region (D–F), anterolateral rod region (G–I), and post‐oral rod region (J–L). Arrows point to skeletogenic cells where the microtubules are enriched in a crescent shape with multiple filaments elongating toward the spicule cavity (D–L). The experiments were done in four biological replicates where the total number of embryos scored is 24. Scale bar in (C, F, I, L) is 10 μm.
	FIGURE 4. Co‐localization of microtubules with the matrix proteins SM30 and SM50 during skeletal formation at 24hpf. Representative images showing sea urchin embryo at 24hpf stained with β‐Tubulin (green), the skeletogenic cell marker 6a9 (blue) and SM30 (red, A–J) or SM50 (red, K–S). Throughout the figure, arrows point to skeletogenic cells where the spicule matrix proteins are enriched in a crescent shape around the nucleus, probably, the Golgi apparatus. (A, F) Different focal plains of the same embryo. (B–E) Zoom‐in into the mineral‐free pseudopodia cable area marked with white rectangle in (A). Arrowheads point to the pseudopodia cable with minimal SM30 signal. (G–J) Zoom‐in into the mineralized spicule area rectangle marked in (F), arrowheads point to the spicule cavity. (L–O) Zoom into the pseudopodia cable area marked with white rectangle in (K). Arrowheads point to the mineral‐free pseudopodia cable where SM50 is enriched. (P–S) Zoom into a mineralized spicule area stained with SM50. White arrowheads point to the spicule cavity where β‐Tubulin and SM50 signals overlap. The experiments were done in two biological replicates and the total number of embryos scored for SM30 is 18 and 33 for SM50. Scale bar in (A, B, F, G, K, L, P) is 10 μm.
	FIGURE 5. SM30 expression overlaps with the microtubule filaments in the skeletogenic lineage at 48hpf. Representative images showing sea urchin embryos stained with SM30 antibody (red), β‐Tubulin (green), and the skeletogenic cell marker 6a9 (blue) at 48hpf. (A–D) Whole embryo images. (E–H) Zoom‐in into body‐tip area marked by a rectangle in (A). (I–L) Zoom‐in into the body‐back area marked in (A), from a different embryo. (M–P) Zoom‐in into the tip of the post‐oral rod marked by a rectangle in (A). (Q–T) Zoom‐in into the anterolateral rod area marked by a rectangle in (A). Arrows point to skeletogenic cells where SM30 is enriched at the Golgi apparatus. White arrowheads point to the mineralized spicule where SM30 signal is high. Yellow arrowheads point to regions where SM30 signal is detected along the microtubule filaments that elongate from the Golgi apparatus toward the spicule. The experiments were done in three biological replicates where the total number of embryos scored is 30. Scale bar in (D) is 50 μm and (E, I, M, Q) is 10 μm.
	FIGURE 6. SM50 expression overlaps with the microtubule filaments within the skeletogenic lineage at 48hpf. Representative images showing sea urchin embryo stained with SM50 antibody (red), Tubulin‐β (green) and the skeletogenic cell marker 6a9 (blue) at 48hpf. (A–D) Whole embryo images. (E–H) Zoom‐in into the body‐tip rectangle in (A). (I–L) Zoom‐in into the body‐back rectangle in (A). (M–P) Zoom‐in into the post‐oral rod marked in (A). Arrows point to skeletogenic cells where SM50 is enriched at the Golgi apparatus. White arrowheads point to the mineralized spicule where SM50 signal is high. Yellow arrowheads point to regions where SM50 signal is detected along the microtubule filaments that elongate from the Golgi apparatus toward the spicule. The experiments were done in two biological replicates where the total number of embryos scored is 12. Scale bar in (A, E, I, M) is 10 μm.
	FIGURE 7. pFAK coats the spicule and overlaps with the edges of the microtubule filaments at 24hpf and 48hpf. pFAK. (A–H) Representative images showing sea urchin embryos stained with pFAK antibody (red), β‐Tubulin (green), and the skeletogenic cell marker 6a9 (blue) at 24hpf. (A–D) Mineralized grain area, where the mineral membrane is coated with active FAK (arrowhead) surrounded by thin microtubule layer. Arrows point to skeletogenic cells where microtubule filaments elongate from around the nucleus to the cell cortex. (E–H) The triradiate spicule area at 24hpf. The spicule membrane is coated with pFAK and the edges of the microtubule overlap with pFAK stain (arrowheads). Arrows point to skeletogenic cell where microtubule filaments elongate toward the spicule. The experiments were done in two biological replicates where the total number of embryos scored is 36. Scale bar in (A, D, E) is 10 μm. (I–P) Representative images showing a sea urchin larva stained with pFAK antibody (red) and β‐Tubulin (green) at 48hpf. (I, M) Whole embryo. (J–L) Zoom‐in into the body‐tip rectangle marked in (I). (N–P) Zoom‐in into the post oral‐tip rectangle marked in (M). White arrowheads point to the mineralized spicule where pFAK signal is high. White arrows point to skeletogenic cells extending microtubule filaments from around the nucleus into the spicule. Yellow arrowheads point where the elongated microtubule filaments overlap with pFAK. The experiments were done in two biological replicates where the total number of embryos scored is 17. Scale bar in (I, M) is 50 μm and in (L, P) is 10 μm.
	FIGURE 8. Proposed model of microtubule structure and function throughout sea urchin larval skeletogenesis. (A) An illustration of a 25hpf sea urchin embryo with skeletogenic cells marked in blue, the biomineral marked in light orange. (B) An enlargement of the triradiate spicule area showing the skeletogenic cells around the spicule with nuclei marked in white, and the Golgi apparatus marked in pink. At the Golgi apparatus matrix proteins SM50 (marked in yellow) and SM30 (marked in purple) are being sorted and packed inside vesicles. Around the spicule FAK is activated (marked in red). The microtubule filaments elongate from perinuclear region into the biomineralization compartment in all the skeletogenic cells, and the microtubule filaments are anchored around the outer membrane of the spicule. In the region of the mineral‐free pseudopodia, microtubule filaments transport vesicles bearing SM50 from the Golgi into the pseudopodia cable while SM30 is only found in the Golgi. At the growing tip of the spicule, both SM30 and SM50 are trafficked on microtubules from the Golgi apparatus to the spicule outer membrane. (C) Illustration of a sea urchin embryo at 48hpf, similar color codes to A. (D) An enlargement of the body tip area. The microtubule filaments elongate from perinuclear region into the biomineralization compartment, possible mediating the transport of SM30 and SM50 bearing vesicles. The density of the microtubule filaments appears to higher at cells near the growing tips compared to cells at the back of the rods. Image courtesy of Shanduo Chen.