Myosin-1c couples assembling actin to membranes to drive compensatory endocytosis

Abstract

Compensatory endocytosis follows regulated exocytosis in cells ranging from eggs to neurons, but the means by which it is accomplished are unclear. In Xenopus eggs, compensatory endocytosis is driven by dynamic coats of assembling actin that surround and compress exocytosing cortical granules (CGs). We have identified Xenopus laevis myosin-1c (XlMyo1c) as a myosin that is upregulated by polyadenylation during meiotic maturation, the developmental interval that prepares eggs for fertilization and regulated CG exocytosis. Upon calcium-induced exocytosis, XlMyo1c is recruited to exocytosing CG membranes where actin coats then assemble. When XlMyo1c function is disrupted, actin coats assemble, but dynamic actin filaments are uncoupled from the exocytosing CG membranes such that coats do not compress, and compensatory endocytosis fails. Remarkably, there is also an increase in polymerized actin at membranes throughout the cell. We conclude that XlMyo1c couples polymerizing actin to membranes and so mediates force production during compensatory endocytosis.

Figure 1. XlMyo1c Is Specifically Upregulated by Polyadenylation during Meiotic Maturation

(A) Poly(A)⁺ northern blots of six myosins and γ-actin from the six stages of oogenesis (1–6; “Oogenesis”) and the egg following meiotic maturation (E; “MM”). Levels of XlMyo1c Poly(A)⁺ RNA increase during meiotic maturation, while levels for all of the other myosins and γ-actin sharply decrease during the same interval (compare 6 to E). Equal amounts of RNA were loaded in each lane. (B) Poly(A)⁺ and total RNA before (6) and after (E) meiotic maturation for XlMyo1c, XlMyo2a, and γ-actin. Total RNA levels do not change for any message, while poly(A)⁺ levels drop sharply for XlMyo2a and γ-actin. In contrast, poly(A)⁺ levels increase for XlMyo1c. Thirty oocyte equivalents of RNA were loaded for each lane. (C) Immunoblot comparison of protein levels before (6) and after (E) meiotic maturation for XlMyo1c, XlMyo2a, and γ-actin. XlMyo1c levels increase during meiotic maturation, while those of XlMyo2a and γ-actin do not. (D) The complete coding sequence for XlMyo1c. The ATP-binding region (dark underline) and actin-binding region (dashed underline) are indicated, as are the three IQ motifs (dark underline, gray text). (E) Schematic diagram of XlMyo1c. (F) Alternative 3′UTRs for XlMyo1c. Both 3′UTRs include nuclear polyadenylation signals (PolyA) but only XlMyo1c(A) has the cytoplasmic polyadenylation element (CPE) necessary to direct polyadenylation during meiotic maturation.

Figure 2. XlMyo1c Is Recruited to Exocytosing CGs upon Egg Activation

(A) Single plane images of eGFP-XlMyo1c (green) and F-actin (red) in oocytes, eggs, and activated eggs. Surface views show XlMyo1c at the PM overlying F-actin microvilli. Upon egg activation, views just below the PM show XlMyo1c is recruited to exocytosing CGs, along with actin. (B) Z sections of a prick-activated egg. In regions far from the prick site, XlMyo1c completely surrounds CGs that do not yet have actin coats. At intermediate distances from the prick site, exocytosing CGs are partially enclosed by actin coats (arrowheads). Near the prick site, exocytosing CGs are completely enclosed by actin coats. (C) Z sections of endogenous XlMyo1c (red; 1C) and F-actin (green; FA) in activated eggs. XlMyo1c localizes to the PM and exocytosing CGs. (D) Immunoblot analysis of cortical (pel) and cytoplasmic (sup) fractions from Xenopus oocytes. Cortical fractions are enriched in cell-surface proteins and CG lectins, while most of the actin is cytoplasmic. Both endogenous and exogenous XlMyo1c are enriched in the cortical fraction. (E) Time-lapse images from a single plane just beneath the PM, showing eGFP-XlMyo1c (green) before and after egg activation by IP₃ uncaging in the presence of extracellular TR-dextran (red) to reveal exocytosing CGs. Within 3 s of uncaging, CGs exocytose (arrowhead), become surrounded by eGFP-XlMyo1c, and are then compressed. See Movie S1. (F) Z section, time-lapse images of eGFP-XlMyo1c recruitment to exocytosing CGs that are then compressed. (G) Single plane time-lapse images showing eGFP-XlMyo1c (green) and actin (red) before and after activation by IP₃ uncaging. eGFP-XlMyo1c is recruited before actin. Time is in min:sec; bars are 5 µm.

Figure 3. Neck and Tail Constructs of XlMyo1c Are Recruited to Exocytosing CGs

(A) Schematic diagram of eGFP-tagged XlMyo1c constructs. (B) Time-lapse images, from a single plane just below the PM, showing eGFP-HIQ, eGFP-IQT, eGFP-T, and eGFP-IQ before and after egg activation by IP₃ uncaging. While eGFP-HIQ remains cytoplasmic following activation, eGFP-IQT, eGFP-T, and eGFP-IQ are recruited to exocytosing CGs (arrowheads). Time is in min:sec; bar is 5 µm.

Figure 4. XlMyo1c Disruption by Dominant-Negative IQT Expression Perturbs Actin/Membrane Events of Egg Activation

(A) Single plane images of eGFP-HIQT signal at the PM showing fluorescence decreases with increasing amounts of IQT coexpression. (B) Quantification of decreasing eGFP-HIQT signal at the PM with coexpression of IQT. Results are mean ± SEM; n = 3 independent experiments. Asterisks indicate p < 0.05. (C) Quantification of oocytes that mature to eggs following progesterone treatment. Expression of IQT has no effect on meiotic maturation relative to controls. (D) Images of propidium-iodide-stained meiotic spindle chromosomes show that IQT expression does not impair spindle rotation during meiotic maturation in that chromosomes are found in classic rosette patterns, parallel to the PM. (E) Z sections of PM showing F-actin in microvilli. IQT expression results in microvilli that are longer than in control cells. (F) Images showing that IQT suppresses cortical contraction in eggs. Calcium ionophore (+Iono.) triggers movement of pigment to animal pole in uninjected (No inj.) and HIQT-injected cells but not in IQT-injected cells. Coexpression of HIQT rescues the effects of IQT. (G) Quantification of cortical contraction in eggs. The effects of IQT are dose dependent and are rescued by coexpression of HIQT. Results are mean ± SEM; n = 3 independent experiments. Asterisks indicate p < 0.05. (H) Quantification of oocytes with fertilization envelopes 2 min after calcium ionophore treatment. IQT expression slows exocytosis in a dose-dependent manner, and this slowing is rescued by coexpression of HIQT. Results are mean ± SEM, n = 3 independent experiments. Asterisks indicate p < 0.05. (I) Coomassie-stained gels showing 2 day expression of IQT completely suppresses CG exocytosis in oocytes treated with PMA or eggs treated with PMA or calcium ionophore. Each lane represents CG lectin released from a single oocyte in uninjected (No inj.) and IQT-injected (IQT) cells. (J) Immunoblots of cortical (P) and cytoplasmic (S) fractions showing IQT does not inhibit constitutive exocytosis of Na⁺-K⁺ ATPase or syntaxin-1. While IQT had no effect on exocytosis of these proteins at the PM relative to uninjected controls, progesterone treatment sharply suppressed their exocytosis.

Figure 5. XlMyo1c Disruption by Dominant-Negative IQT Expression Leads to Unrestrained Actin Assembly

(A) Single plane time-lapse images of oocytes injected with either HIQT or IQT and Alexa488-G-actin and then treated with PMA. F-actin comets (arrowheads) are numerous in the IQT but not the HIQT-expressing oocytes. (B) Quantification of the average number of comets appearing in PMA-treated oocytes. IQT significantly increases comet number relative to controls, while coexpression of IQT with HIQT sharply reduces this effect. Results are mean ± SEM; n = 3 independent experiments. Asterisk indicates p < 0.05. (C) Immunoblot of actin in cortical (P) and cytoplasmic (S) fractions shows that IQT increases levels of actin associated with the cortical fraction. This effect is rescued by coexpression of IQT with HIQT. Bars are 10 µm.

Figure 6. XlMyo1c Disruption by a Morpholino Perturbs Actin/Membrane Events of Egg Activation

(A) Immunoblot showing a XlMyo1c morpholino (Xl Myo1c Morpho) prevents the increase in XlMyo1c protein during meiotic maturation, but the mismatch control morpholino (Control Morpho) does not. Tubulin was probed as a loading control. (B) Quantification of cortical contraction in eggs with XlMyo1c morpholino or control morpholino. The XlMyo1c morpholino suppresses cortical contraction. Results are mean ± SEM; n = 3 independent experiments. Asterisk indicates p < 0.05. (C) Quantification of actin comets in oocytes with XlMyo1c morpholino or control morpholino. XlMyo1c morpholino causes a significant increase in actin comets in oocytes. Results are mean ± SEM; n = 3 independent experiments. Asterisk indicates p < 0.05. (D) Coomassie-stained gel showing the XlMyo1c morpholino sharply reduces CG exocytosis in eggs. (E) Coomassie-stained gel showing that cytochalasin D (CytoD) rescues CG exocytosis in IQT-expressing cells (IQT).

Figure 7. XlMyo1c Couples Dynamic Actin to Exocytosing CG Membranes

(A) Time-lapse images from a single plane just below the PM of Alexa488-G-actin (green) in eggs expressing either HIQT or IQT. Eggs were activated by IP₃ uncaging in the presence of extracellular TR-dextran (red) to reveal exocytosing CGs. In HIQT eggs, actin coats rapidly enclose and compress exocytosing CGs (arrowheads). See Movie S2. In IQT eggs, CG exocytosis is slower, as judged by delay in the appearance of dextran. Also, actin coats assemble around exocytosing CGs but fail to compress and eventually dissipate (arrowheads). See Movie S3. (B) Low magnification, 4D time-lapse images of eggs expressing IQT. Uncaging triggers exocytosis; however, CGs are not covered over by actin coats on either the exoplasmic surface (Apical) or the cytoplasmic surface (Basal). See Movie S4 (Apical) and Movie S5 (Basal). (C) High magnification, 4D time-lapse images of eggs expressing either HIQT or IQT. In HIQT eggs, basal views show that the actin coat tracks closely over the cytoplasmic surface of the exocytosing CG, eventually enclosing it. See Movie S6. In IQT eggs, basal views show that the actin coat forms (arrowhead) but is separated from the cytoplasmic surface of the CG (arrows). The coat eventually regresses. See Movie S7. (D) High magnification, 4D time-lapse images of an egg expressing IQT. This tilted z view shows perturbed actin-coat assembly in the form of actin fingers (arrowheads) that partially track along the CG but then extend into the cytoplasm. The fingers grow and shrink over time, but the CG is never completely surrounded by an actin coat. Compression of the incomplete coat causes the CG to squirt out through a region with no coat. (“Out” and “In” indicate the outside and the inside of the cell, respectively.) See Movie S8. (E) High magnification, 4D time-lapse images of eggs expressing either HIQT or IQT. Z views show in HIQT eggs, actin coats extend downward from the PM (arrowheads), enclose the exocytosing CG, and compress upward. See Movie S9. In IQT eggs, while coats start to assemble downward from the PM (arrowheads), they subsequently regress upwards. See Movie S10. (F) Z views from eggs expressing either HIQT or IQT. Only the TR-dextran is shown. In HIQT eggs, exocytosing CGs have broad necks where they fuse to the PM (arrowhead). In IQT eggs, the necks are narrow. Time is in min:sec; bars are 5 µm.