Early morphogenetic events are accomplished by maternal cellular machinery that is developmentally controlled by expression of the zygotic genome (Wieschaus, 1996
). We find that the zygotic gene-product Nullo acts as a developmental switch at cycle 14 and targets the endocytic machinery to cellularize the embryo. Here we assayed endocytic dynamics by following WGA internalization in living embryos and Amph tubulation in fixed embryos. We find that WGA is a general marker for endocytosis, while Amph tubules are more specifically associated with the initial ingression of PM furrows. Several findings support that these Amph tubules are endocytic intermediates: First, their structure is tubular rather than sheet-like, consistent with a role in endocytosis. Second, perturbation of Dynamin, a catalyst of endocytic scission, increases the number of Amph tubules at cellularization furrows. Third, in living nulloX
and Cyto-D treated embryos, WGA is internalized in long, PM-tethered tubules that resemble Amph tubules. Fourth, DPATJ enters Amph tubules in nulloX
embryos, and accumulates at early endosomes. We have, thus, used Amph tubules as quantifiable reporters of endocytosis at furrows.
The membrane furrows that form during the early mitotic cycles regress, while those that form at cellularization stably ingress. We propose that endocytosis is differentially controlled to achieve these distinct morphogenetic events. During the mitotic cycles, metaphase furrows are transient, ingressing only ~5 μm before completely regressing. We find that restrained endocytosis, detected by both WGA internalization and Amph tubules, accompanies the initial furrow ingression that occurs at prophase/metaphase. This is followed by a fast wave of vigorous WGA endocytosis that traverses the embryo surface when metaphase furrows regress at anaphase/telophase. The endocytosis accompanying furrow regression is not associated with high levels of Amph tubules. While this endocytosis may not recruit Amph, we favor a model whereby endocytic scission is more efficient at this time, precluding the capture of tubule intermediates by fixation. Thus, endocytosis at furrow regression may be mechanistically distinct from endocytosis at furrow ingression. It may also be functionally distinct and could even drive furrow regression, as endocytosis adjusts the surface area of both motile and dividing cells (Boucrot and Kirchhausen, 2007
; Traynor and Kay, 2007
). So throughout the early mitotic cycles, alternating and distinct endocytic dynamics are regulated by cell cycle progression and correlate with specific furrow events.
At the onset of cellularization, furrows form in a way that resembles metaphase furrows, but then assemble furrow canals that stabilize the furrow and sustain ingression over ~40 μm. We find that endocytosis, marked by both WGA internalization and Amph tubules, also accompanies the initial ingression of cellularization furrows, but ceases by the time furrows reach 5 μm in length and furrow canals fully assemble. Since cellularization occurs during interphase, cell cycle progression cannot regulate this endocytosis. Instead, zygotic expression of Nullo aids endocytic scission, and this has the effect of limiting membrane dynamics at the tip of the incipient cellularization furrow, so that proteins, including Myosin-2, Septin, and DPATJ, that concentrate there are retained there. As a result, furrow canals assemble and stabilized furrows ingress to cellularize the embryo. Thus, Nullo regulation of endocytic dynamics could promote the developmental transition from transient furrowing that maintains the syncytium to stable furrowing that generates the primary epithelial cell sheet.
Nullo activity facilitates endocytic scission such that budding vesicles are rapidly released from the PM. When scission is impaired some budding vesicles are distended into long Amph tubules that remain persistently tethered to the PM. This phenotype is mimicked when F-actin levels are reduced with Cyto-D, and Nullo regulates cortical F-actin. Thus, we suggest that Nullo aids scission via its regulation of F-actin. How Nullo regulates actin remains elusive as it is a small (213 amino acids), highly basic (pI 11.4), myristoylated protein with no readily identifiable globular domains to suggest interaction partners. Instead, sequence composition and hydropathy character suggest that Nullo is “natively unfolded”, containing 63% disorder promoting amino acids (T, R, G, Q, S, N, P, D, E, and K) over its entire length (Williams et al., 2001
) and a disordered run of 50 consecutive amino acids as predicted by PONDR analysis (L136 - A185). Other disordered proteins have been identified that control F-actin organization, such as MARCKs, MARCKs-related proteins and GAP43 (Arbuzova et al., 2002
; Larsson, 2006
), either by direct interaction with actin or by locally sequestering the actin-regulator PIP2
within the PM. While sequence comparison and lack of characteristic, acidic regions does not suggest that Nullo is MARCKs-related, we find that Nullo interacts with PIP2
in in vitro
binding assays (Sokac and Wieschaus, unpublished data). Nullo may then concentrate PIP2
locally to regulate actin and/or to couple actin to components of the endocytic machinery that also interact with PIP2
at the PM (Di Paolo and De Camilli, 2006
Nullo may aid endocytic scission via F-actin by either active or passive mechanisms. In yeast, F-actin actively drives endocytic scission by exerting polymerization and myosin-based forces to lengthen, and eventually break, the budding vesicle neck (Kaksonen et al., 2003
; Kaksonen et al., 2005
; Sun et al., 2006
). In Drosophila hemocytes and mammalian cells, F-actin also contributes to a late step in endocytosis that just precedes vesicle release, and may be either bud invagination or scission (Kochubey et al., 2006
; Merrifield et al., 2002
; Merrifield et al., 2004
; Yarar et al., 2005
). Additionally, cortical F-actin passively regulates endocytic dynamics by reinforcing the PM and so antagonizing PM deformation. In the case of BAR domain activity, drug mediated reduction of F-actin levels enhances PM tubulation (Itoh et al., 2005
). Reduced levels of cortical F-actin in nulloX
mutants result in the appearance of more Amph tubules. But we find that fewer vesicles are released in living nulloX
embryos, and that Amph tubulation does not expand to regions beyond the furrow, arguing that there is not more endocytosis in mutants. Thus, impaired scission generates the appearance of more tubules, and we find that it takes almost three times longer for budding vesicles to release from the PM in nulloX
versus wild-type embryos. Consistently, dynamin defects enhance PM tubulation in cultured cells and BAR-induced membrane tubulation is antagonized by co-expression of dynamin (Itoh et al., 2005
). So Nullo may regulate a population of F-actin that either actively aids scission, or stiffens the cortex and somehow contributes to endocytosis (ie. if breaking the bud neck is aided by the PM being under cortically-maintained tension).
Our analysis strongly supports that membrane trafficking is differentially controlled at specific sites and times within embryos to achieve distinct morphogenetic events. This was previously suggested for fly embryos by the membrane-labeling analysis of Lecuit and Wieschaus (2000)
, which demonstrated that exocytosis occurs at specific sites along cellularization furrows and so helps establish apical/basal polarity in these cells. Two additional observations were made at that time that are relevant to the results described here: First, in the earlier study membrane labeling of the furrow canal was only possible at very early cellularization. After that time the furrow canal persisted as a stable membrane compartment where no new membrane was either added or taken away. Our data also supports that membrane turnover at the furrow canal region is restricted to the very beginning of cellularization. In fact, we now find that endocytic dynamics are tightly controlled there to establish and/or maintain the concentration of proteins at the furrow canal. Second, in the previous study it was observed that membrane label was cleared from the apical PM, and was suggested that clearing is mediated by endocytosis. At furrow lengths > 5 μm, we also see WGA vesicles moving away from the apical PM. But when our peri-vitelline injections were done with higher lectin concentrations and at furrow lengths < 5 μm, they revealed WGA endocytosis from the tips of incipient furrows. In fixed embryos where spatial resolution is better, Amph tubules clearly extend only from furrow tips. Thus, our analysis shows that in addition to apical endocytosis, local endocytosis also occurs where furrows first ingress.
During cytokinesis in some mammalian cells (Schweitzer et al., 2005
) and in plants (Dhonukshe et al., 2006
), membrane endocytosed at sites remote from the furrow is later delivered to the division plane via the endocytic pathway. At cellularization furrows in the fly embryo the exocytosis of membrane derived from recycling endosomes suggests a similar pathway. In these cases, endocytosis from one site can provide a store of membrane to feed growth somewhere else. But our observation that endocytosis occurs at the furrow itself is counterintuitive since endocytosis would be expected to reduce surface area while furrow ingression requires surface expansion. Nonetheless, there are now several reports that endocytic proteins including Clathrin, Clathrin adaptor-2 and Dynamin concentrate in cytokinesis furrows (Albertson et al., 2005
). In addition, endocytosis has been directly visualized at furrows in dividing Zebrafish embryos (Feng et al., 2002
), cultured cells (Sweitzer and Hinshaw, 1998
; Warner et al., 2006
) and fission yeast (Gachet and Hyams, 2005
), although the function of this endocytosis remains unclear.
We report that endocytosis occurs at the tips of both metaphase and cellularization furrows when the furrows are first ingressing, suggesting that it confers some temporally and spatially specific function. Both the PM and actin are significantly remodeled at these sites as furrows form. At the onset of cellularization in particular, the F-actin/Myosin-2 furrow canals are assembling at this place and time. Actin remodeling is intimately coupled to endocytosis in other cell types (Smythe and Ayscough, 2006
): Endocytic proteins control actin dynamics and actin binding proteins are required for endocytosis. Also, endocytic and actin binding proteins are regulated by the same phosphoinositide pools at the PM (Di Paolo and De Camilli, 2006
). It follows that local endocytosis could influence local actin organization during furrow formation in fly embryos. Here we show that actin conversely provides developmental regulation of endocytic dynamics. This analysis leads us to speculate that the coupled regulation of actin and endocytosis can effectively coordinate actin/PM remodeling to drive furrow dynamics, and so shape cells during morphogenesis.