F-actin asymmetry and the endoplasmic reticulum-associated TCC-1 protein contribute to stereotypic spindle movements in the Caenorhabditis elegans embryo

Abstract

The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα-GPR-LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα-GPR-LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division.

FIGURE 1:

Localization of the LIN-5 complex at the cell periphery does not require an intact actin or microtubule cytoskeleton. (A) perm-1(RNAi) embryos treated with the microtubule-depolymerizing drug nocodazole or with solvent only (1% DMSO, Control). Embryos were stained for LIN-5, tubulin, and DNA (DAPI). Note that microtubules are severely depolymerized in nocodazole-treated embryos, but that LIN-5 still localizes to the cell periphery (arrows). (B) perm-1(RNAi) embryos treated with the actin-depolymerizing drug latrunculin A, cytochalasin D, or with 1% ethanol (control for cytochalasin D). Embryos were probed with antibodies for LIN-5 and actin. DAPI was used to visualize DNA. Note that LIN-5 is present at the cell periphery of both control and latrunculin A–treated embryos (arrows). (C) Time-lapse images of YFP::GPR-1 embryos permeabilized with perm-1 RNAi and treated with solvent only (Control), latrunculin A, or a combination of latrunculin A and nocodazole. Note that even simultaneous disruption of microtubules and actin does not prevent GPR-1 membrane localization (bottom two sets of panels).

FIGURE 2:

Actin inhibits pulling forces in the anterior. Centrosome movements are depicted for a control embryo (A), lin-5(ev571ts) embryo (B), cytochalasin D–treated embryo (C), and a lin-5(ev571ts) embryo treated with cytochalasin D (D). (E) Maximum amplitudes from spindle pole oscillations during anaphase. Values of the anterior and posterior spindle poles of indicated embryos are shown. Average values (± SD, n ≥ 6). (F) Position of the spindle poles during mitosis from nuclear envelope breakdown on in control and cytochalasin D–treated embryos (expressed as % egg length (x-axis) over time in seconds (y-axis), ± SD; control: n = 8; cytochalasin D: n = 9.

FIGURE 3:

Actin inhibits anterior pulling force generation. (A) Time-lapse series of a mitotic spindle in a control embryo, in which the midzone spindle is severed at anaphase onset (arrow). Note that the posterior spindle pole moves with a higher velocity than the anterior spindle pole. (B and C) Kymographs of mitotic spindles from a control (B) and a cytochalasin D–treated embryo (C) after midzone severing. Kymographs are taken from a single longitudinal line across the mitotic spindle. The spindle poles are visualized by GFP::TBB-2. Compared with control embryos (B), both spindle poles move with high velocities after midzone ablation in cytochalasin D–treated embryos (C). (D) Spindle pole peak velocities after severing the spindle midzone at anaphase onset with a UV laser. Values are shown for embryos treated with cytochalasin D, latrunculin A, or solvent only (control Cyto. D: egg buffer + 1% EtOH; control Lat. A : egg buffer plus 1% DMSO). Pole velocities in the latter control embryos were slightly higher than normal (average values ± SEM). Anterior pole velocity is significantly increased in the presence of cytochalasin D (p < 0.0007 for solvent compared with cytochalasin D; p < 0.3 for solvent compared with latrunculin A. Pole velocities in the latter control embryos were slightly higher than normal. n ≥ 10 embryos for each treatment.

FIGURE 4:

TCC-1 inhibits cortical pulling forces, possibly through inhibition of GPA-16 localization at the plasma membrane. (A) Illustration of TCC-1 domain structure. (B and C) Transverse spindle pole movements during anaphase in (B) a normal (N2) and (C) tcc-1(RNAi) embryo. (D) Maximum amplitude of the anterior and posterior spindle pole during anaphase in N2 and tcc-1(RNAi) embryos. Average values (± SD; n ≥ 10). (E) Spindle pole positioning in N2 and tcc-1(RNAi) embryos, indicated by the position of the centrosomes, expressed as % egg length (x-axis) over time in seconds (y-axis). (F) Embryonic lethality observed in the depicted strains treated with RNAi for gfp or tcc-1 at 20°C. Average values (± SD) from experiments performed in duplicate with three hermaphrodites for each condition. (G) Left–right axis reversal of indicated strains at 20°C, treated with RNAi for either gfp or tcc-1. Average values (± SD) from 65 to 112 animals were scored for each condition. (H) Spindle pole peak velocities after severing the midzone spindle at anaphase onset with a UV laser. Values are shown for normal (N2) and tcc-1(RNAi) one-cell embryos. Average values are indicated for each pole (± SEM; N2: tcc-1: n = 17; RNAi: n = 31). (I) Control and tcc-1(RNAi) embryos stained for GPA-16 and DNA (DAPI). Note that GPA-16 localization is enriched at the plasma membrane in tcc-1(RNAi) embryos (arrows). The right panel shows a quantification of cortical GPA-16 enrichment measured at the contact between the AB and P2 cell. Average values are indicated (± SD; n = 14).

FIGURE 5:

TCC-1 localizes to the ER. (A) One-cell embryo at anaphase onset, and four-cell embryo in interphase. Note that TCC-1::mCherry associates with the mitotic spindle (left panel, arrowhead), nuclear envelope (right panel, arrow), and plasma membrane (left panel, arrow). The organization is more sheet-like in mitosis and becomes dispersed at the end of mitosis. (B) Localization of TCC-1::mCherry and the ER marker SP12::GFP in early embryos. Note the overlap in localization and close association of TCC-1::mCherry and SP12::GFP with the meiotic spindle and nearby cortex (top, arrow). The organization changes from membranous with foci in meiosis and mitosis to dispersed in interphase. (C) Extensive clustering of the ER in tcc-1(RNAi) embryo (right, indicated by arrows). In a wild-type embryo, some clusters can be detected in mitosis (left).

FIGURE 6:

TCC-1 contributes to translocation of the meiotic spindle toward the cortex. Depicted are still images from time-lapse recordings of normal (Control), tcc-1(RNAi), and unc-116(RNAi) embryos. In control embryos, the meiotic spindle stays in close proximity to the anterior cortex. After spindle shortening, a rotation causes the spindle to be positioned perpendicular to the cortex (arrowhead). In tcc-1(RNAi) embryos, the meiotic spindle becomes transiently displaced from the cortex (arrow, second row), as in unc-116(RNAi) embryos (arrow, bottom row). In some tcc-1(RNAi) embryos, the meiotic spindle appears to drift or tumble away from the anterior end (arrow, third row). See Supplemental Movies S1–S11 for further details.