Polarization of the endomembrane system is an early event in fucoid zygote development

Abstract

Background: Fucoid zygotes are excellent experimental organisms for investigating mechanisms that establish cell polarity and determine the site of tip growth. A common feature of polarity establishment is targeting endocytosis and exocytosis (secretion) to localized cortical domains. We have investigated the spatiotemporal development of endomembrane asymmetry in photopolarizing zygotes, and examined the underlying cellular physiology.

Results: The vital dye FM4-64 was used to visualize endomembranes. The endomembrane system preferentially accumulated at the rhizoid (growth) pole within 4 h of fertilization. The polarized endomembrane array was initially labile and reoriented when the developmental axis changed direction in response to changing light cues. Pharmacological studies indicated that vesicle trafficking, actin and microtubules were needed to maintain endomembrane polarity. In addition, endocytosis required a functional cortical actin cytoskeleton.

Conclusion: Endomembrane polarization is an early event in polarity establishment, beginning very soon after photolocalization of cortical actin to the presumptive rhizoid site. Targeting of endocytosis and secretion to the rhizoid cortex contributes to membrane asymmetry. We suggest that microtubule-actin interactions, possibly involving microtubule capture and stabilization at actin-rich sites in the rhizoid, may organize the endomembrane array.

Figure 1

Endomembrane asymmetry in polarizing zygotes. (a) Zygotes 6 h AF were double labeled with fluorescent microspheres (green) to visualize polar adhesive (arrowhead) and FM4-64 (red) to visualize endomembrane distribution. Arrow indicates direction of orienting light vector and scale bar = 50 μm. Adhesive and endomembranes accumulate at rhizoid (shaded) pole. (b) Time course of photolocalization of adhesive and endomembranes. Y axis indicates percent of zygotes with polar adhesive or polar endomembrane distribution. Over 100 zygotes were scored for each time point and the experiment was repeated three times. Bars are standard errors.

Figure 2

Endomembrane accumulation marks the rhizoid pole. Vital staining of a field of 6 zygotes with FM4-64 during photopolarization (a, 8 h AF) and following germination (b, 20 h AF). Each zygote germinated at the position previously marked by brightest FM4-64, except for the cell indicated by an asterisk, which had uniform labeling at 6 h and did not germinate. Arrow indicates direction of orienting light vector and scale bar = 100 μm.

Figure 3

Endomembrane asymmetry can be reoriented. Zygotes were grown in unilateral light until 6 h AF (L1), at which time the light direction was reversed (L2). Zygotes were labeled for 30 min at hourly intervals beginning at 6 h AF and scored as having polar adhesive or endomembrane in accordance with L1 or L2. Zygotes with uniform labeling were not scored. (a) Adhesive deposited in accordance with L1 (arrow) was not resorbed and a second zone of polar adhesive (arrowhead) was deposited in accordance with L2. Bar = 50 μm. (b) Adhesive deposition reversed following light reversal. Note that filled squares represent adhesive localization with L1 and open circles represent zygotes with two patches of polar adhesive. (c) Endomembrane localization with L1 (filled squares) decreased with time after light reversal and localization with L2 (open circles) increased. Over 100 zygotes were scored for each time point and the experiment was repeated three times. Bars are standard errors.

Figure 4

Endocytosis contributes to polar endomembrane labeling. A 6 h-old zygote was labeled for 2 min and the spatial distribution of endocytosis was immediately imaged by confocal microscopy. Image is a single optical section near the median plane demonstrating preferential FM4-64 uptake at the rhizoid pole. Arrow indicates direction of polarizing light and bar = 50 μm.

Figure 5

Endocytosis is inhibited by actin disruption. Zygotes 6 h AF were treated for 30 min with DMSO (a) or 30 nM Lat B (b) and then labeled with FM4-64 for 30 min in the presence of drug. Diffuse cortical fluorescence in (b) is primarily due to out-of-focus labeling of plasma membrane. Bar = 50 μm.

Figure 6

Actin disruption accelerates loss of polar endomembrane labeling. At 6.5 h AF, zygotes on coverslips in Petri dishes were labeled for 30 min followed by 3 × 5 min rinses in ASW to wash out FM4-64. Zygotes were immediately scored for initial endomembrane polarity. Either 0.06% DMSO or 30 nM Lat B was then added to a dish and endomembrane polarity was again scored 1 and 2 h later on separate coverslips. At the time indicated by the arrow (2 h after FM4-64 washout), the media were spiked with FM4-64. Y axis indicates percent of zygotes with polar endomembrane distribution. Over 100 zygotes were scored for each point and the experiment was repeated three times. Bars are standard errors.

Figure 7

Perturbation of microtubule dynamics or membrane trafficking alters endomembrane distribution. (a) Experimental design. Zygotes on coverslips in Petri dishes were treated chronically with drug beginning at 6 h AF. At 30 min intervals thereafter a separate coverslip was labeled with FM4-64 for 30 min. Arrows at the end of the FM4-64 labeling period indicate time at which zygotes were scored for endomembrane asymmetry. (b-d) Time course of changes in endomembrane asymmetry following treatment with 5 μM paclitaxel (b), 5 μM oryzalin (c), or 5 μg/ml BFA (d). Y axis indicates percent of zygotes with polar endomembrane distribution. Each experiment was repeated three times and over 100 zygotes were scored for each time point. Bars are standard errors. Images are zygotes treated with 0.05% DMSO (e), paclitaxel (f), oryzalin (g) or BFA (h). Bar = 50 μm