Background

Membrane protrusions are specialized cellular structures that perform diverse functions, including environmental sensing, generation of traction forces during cell migration, and mediation of intercellular communication. Beyond their roles in normal development and cellular homeostasis, these structures are also implicated in disease progression [1]. In developing multicellular organisms, membrane extensions enable long-range communication between cells, which is crucial for coordinating complex morphogenetic processes.

Membrane protrusions exhibit substantial structural and functional diversity and are described by various terms, such as cytonemes, tunneling nanotubes (TNTs), membrane nanotubes, and microtubule-based nanotubes (MT-nanotubes) [2,3]. While these structures can connect distant cells, they differ in cytoskeletal composition and function. Cytonemes are predominantly actin-based extensions that facilitate the targeted delivery of signaling molecules to neighboring cells [4]. In contrast, nanotubes are generally microtubule-based and support a broader range of cellular activities, including the transport of vesicles, organelles, signaling molecules, and survival factors [5,6]. Long, narrow protrusions (typically less than 1.5 μm wide) that span several cell diameters are commonly classified as MT-nanotubes [7]. When these nanotubes form open connections between cells, they enable direct cytoplasmic exchange. While this connectivity can support normal physiological communication, it can also be exploited by pathogens or cancer cells to promote survival and disease progression.

Closed membrane nanotubes, on the other hand, often function in paracrine or juxtacrine signaling and play essential roles in tissue morphogenesis [8]. For example, in the Drosophila male germline, closed nanotubes mediate communication between germline stem cells and hub cells, maintaining stem cell identity [9]. Similarly, actin-based protrusions coordinate myoblast fusion during muscle development, and cytonemes facilitate long-range morphogen signaling during Drosophila wing and eye development [10,11]. Despite the diversity, the mechanisms underlying nanotube formation and function remain poorly understood. Drosophila micropyle development provides a useful model to study how thin membrane protrusions contribute to organ morphogenesis. The micropyle is a tubular structure at the anterior end of the eggshell that allows sperm entry during fertilization [12]. From stage 12 onward, a thin protrusion from the polar cells extends into the developing micropyle and generates an open channel in the eggshell that serves as an entry point for fertilizing sperm (Figure 1A–E) [13].

Figure 1. Schematic of stage-wise tubular morphogenesis of the micropyle at late oogenesis. (A) By stage 10, the border cell (BC) cluster has completed its migration and reaches the oocyte, while the centripetal follicle cells (CFCs) begin migrating inward from both the dorsal and ventral sides of the egg chamber. These CFCs move to enclose the anterior region of the oocyte, creating a partition between the nurse cells (NCs) and the oocyte. (B) At stage 11, CFC migration is completed, and they initiate the early steps of tubular morphogenesis of the micropyle. Concurrently, polar cells (PCs), located centrally within the BC cluster, begin to form a protrusion directed toward the oocyte surface, marking the tip of the developing micropyle. (C–E). From stage 13 onward, this protrusion elongates into a narrow, thin extension that pierces the forming tubular structure, contributing to the creation of an open channel that serves as the conduit for fertilizing sperm. In the schematic representation, NCs are depicted in pastel purple, the oocyte in pastel magenta, BCs in green, PCs in yellow, CFCs in blue, the vitelline membrane (VM) in sky blue, and the complete extra chorionic structure known as micropyle (MP). (Images were modified from Acharjee et al. [13].)

A major challenge in studying these tubular structures is that they are extremely thin, fragile, and dynamic. Fixed-tissue imaging provides useful structural information but captures only static snapshots, making it difficult to resolve protrusion formation, extension, remodeling, and interactions with surrounding cells. In addition, delicate membrane structures may be distorted or lost during fixation. Thus, live-cell imaging offers a key advantage by enabling direct visualization of protrusion dynamics in real time and revealing the cellular events that drive morphogenesis.

Here, we describe an optimized live-cell imaging protocol for visualizing the polar cell protrusion during micropyle morphogenesis in Drosophila egg chambers. The protocol includes methods for accurate staging and fine dissection of egg chambers, which are essential for isolating intact samples at the appropriate developmental window. We also optimized the ex vivo culture conditions to maintain egg chamber viability for extended imaging periods, allowing continuous observation of this dynamic event. In addition, imaging parameters were refined to minimize phototoxicity and sample heating during prolonged acquisition, preserve tissue health, and prevent imaging-induced artifacts. These optimizations improve sample stability and image quality, enabling reproducible acquisition of high-resolution datasets across the complete morphogenetic process. The protocol is also compatible with pharmacological treatments, providing a practical platform for functional perturbation experiments in isolated egg chambers. Overall, this method offers a robust and accessible approach for studying dynamic membrane protrusions in living tissue and, with minor modifications, may be adapted to investigate related developmental processes in other insect systems.