Background

An essential aspect of cell homeostasis is the maintenance of various cellular components, including proteins and lipids, and even the integrity of entire organelles. One such organelle is the centrosome, a conserved eukaryotic structure that plays a crucial role in several cellular processes. These include the regulation of cell adhesion, polarity, and mobility during interphase, as well as the assembly of the mitotic spindle, mainly composed of microtubules (MTs) [1,2]. Centrosomes serve as the primary microtubule-organizing centers (MTOCs) in proliferating eukaryotic cells. Each centrosome is composed of two centrioles—MT-based cylindrical structures surrounded by a multiprotein matrix known as the pericentriolar material (PCM) (Figure 1A). The PCM contains factors essential for MT nucleation and anchoring, also being critical for centriole biogenesis [3]. In cycling cells, the number of centrosomes is tightly controlled. Centrosomes are typically duplicated once per cell cycle, with duplication coupled to DNA replication, during the S phase [4]. In some cell types, during interphase, the centrosome migrates to the cell cortex, promoting the growth of an MT-based axoneme to form cilia or flagella [4]. Deregulation of centrosomal components leads to a wide range of diseases, many of which are incompatible with life.

Historically, centrosomes have been regarded as exceptionally stable structures [5,6]. However, in some differentiating cell types, these structures can be inactivated (i.e., lose their MTOC activity) or even disappear, as seen in epithelial cells, muscle cells, neurons, and oocytes [7–9]. Advances in various technological approaches have uncovered the major conserved components and pathways regulating centrosome duplication and maturation into fully functional MTOCs. However, much less is known about the components involved in the maintenance of mature centrosomes or centrioles, the pathways that regulate their inactivation or elimination, and how deregulation of these pathways may contribute to disease [10]. Hyperactive MTOC function at centrosomes has been linked to epithelial cancers and invasive tumor cell behavior [11–15]. Additionally, centrosome inactivation has been proposed as a strategy employed by cancer cells to silence supernumerary centrosomes, which would otherwise lead to cell death [16]. Therefore, elucidating the mechanisms that regulate centrosome and centriole stability is crucial not only for understanding the pathways of centrosome inactivation and/or elimination in post-mitotic and differentiating cells but also for uncovering how deregulation of these processes may contribute to cancer and other diseases.

Studying centrosome maintenance poses experimental challenges and limitations. Experimental setups must ensure that any manipulation (loss- or gain-of-function, such as in vivo RNAi or ectopic expression) is performed only after centrosomes have been fully assembled and that such manipulations do not affect centriole biogenesis. This requires tools for acute spatial-temporal manipulation, which are often unavailable or difficult to implement in many systems. Additionally, depending on the model system used, simultaneously manipulating (depleting or over-expressing) two or more components in vivo can be very challenging and time-consuming.

Taking into account these limitations, we provide a protocol that uncouples centriole biogenesis from maintenance in Drosophila melanogaster cultured cells (DMEL-2). By using this protocol, we have successfully identified components required for the maintenance of centrosome integrity [17,18]. That knowledge allowed us to generate tools to validate the function of such components in vivo [17,18]. In this assay, cells are arrested in S phase using aphidicolin (APH) and hydroxyurea (HU), which stall DNA replication. Unlike some commonly used human cultured cell lines, which allow for multiple rounds of centriole reduplication under S phase arrest conditions [19–21], Drosophila cultured cell lines do not exhibit this behavior. This distinction allows for the separation of centrosome duplication from centrosome maintenance. Moreover, Drosophila cultured cells are easy to culture and manipulate, and the addition of long double-stranded RNAs (dsRNA) enables the depletion of different proteins individually or simultaneously by RNAi. Therefore, following treatment with HU and APH, the number of centrioles is not affected by pathways involved in centriole biogenesis. This allows for the investigation of the effects of different experimental manipulations on centrosome stability.