Giuliano Callaini

The centriole is well recognized for its ninefold symmetry, with nine microtubule blades arranged in a barrel-like structure. However, these blades adopt diverse architectures across evolution, ranging from microtubule singlets to triplets, and the mechanisms governing this variation and its biological significance remain largely elusive. Here we found that in Drosophila male germline cells, routine conversion of microtubule doublets to triplets is impaired in the absence of Ana1. The Ana1 N-terminal region localizes adjacent to microtubule doublets and promotes modest elongation, whereas the C-terminal region extends outward and is sufficient to support C-tubule assembly. Ana1 recruits centrobin to centrioles via its C-terminal region, and targeted recruitment of centrobin to centrioles restores C-tubule formation in an Ana1-deficient background. Restoring Ana1 expression at different developmental stages reveals that the integrity of triplet microtubules is critical for male fertility. Together, these findings identify a regulator of centriolar doublet-to-triplet conversion and provide insights into the biological significance of this process.

Stick insect sperm exhibit a remarkable modification of the basal body, characterized by the expansion of the proximal region and the incorporation of amorphous, unstructured material. This atypical architecture parallels transformations observed in mammalian sperm, where the distal centriole transitions into splayed microtubules of variable length. Despite their structural irregularity, these modified centrioles enable zygotic centrosome formation, likely relying on a large permissive recruitment domain of specific proteins. Therefore, atypical centrioles may represent a sort of assembly platform, rather than providing a defined basal scaffold as observed in other systems. Basal stick insects possess canonical cylindrical centrioles, whereas higher stick insects show increasing basal body complexity. This trend mirrors mammalian sperm evolution, suggesting structural parallels of the basal bodies, presumably enhancing mechanical support to the sperm neck during flagellar motion.

Centrioles are remarkably stable organelles that play a main role in the assembly of centrosomes and ciliary structures. However, there are several differentiated tissues that must eliminate their centrioles to avoid centrosome formation and improper cell proliferation. Therefore, centriole elimination represents an important process in many organisms to ensure successful cell differentiation and maintenance of tissue homeostasis. In this review, we analyzed centriole inactivation and elimination in various Drosophila cell types in relation to the dynamics of the pericentriolar material.

The PACS (phosphofurin acidic cluster sorting protein) proteins are membrane trafficking regulators, required for maintaining cellular homeostasis and preventing disease states. Mutations in human PACS1 and PACS2 cause human neurodevelopmental disorders, characterized by epileptic seizures and neurodevelopmental delay. In vertebrates, functional analysis of PACS proteins is complicated by the presence of two paralogues which can compensate for the loss of each other. Here, we characterize the unique fly homologue of human PACS proteins. We demonstrate that Drosophila PACS (dPACS) is required for cell division in dividing spermatocytes and neuroblasts. In primary spermatocytes, dPACS colocalizes with GOLPH3 at the Golgi stacks and is essential for maintaining Golgi architecture. In dividing cells, dPACS is necessary for central spindle stability and contractile ring constriction. dPACS and GOLPH3 proteins form a complex and are mutually dependent for localization to the cleavage site. We propose that dPACS, by associating with GOLPH3, mediates the flow of vesicle trafficking that supports furrow ingression during cytokinesis. Furthermore, loss of dPACS leads to defects in tubulin acetylation and severe bang sensitivity, a phenotype associated with seizures in flies. Together our findings suggest that a Drosophila PACS disease model may contribute to understanding the molecular mechanisms underpinning human PACS syndromes.

Wolbachia are obligate intracellular alphaproteobacteria that enhance their spreading by altering the reproductive mechanisms of several invertebrates. Among the reproductive alterations, Wolbachia also causes cytoplasmic incompatibility that leads to embryo death when infected males are crossed with uninfected females, thus selecting infected females. However, the presence of Wolbachia has important fitness costs and infected Drosophila simulans males produce less sperm than their uninfected counterparts. Such sperm suffer, indeed, of some structural alterations that hinder their proper function. We took advantage of the fact that several sperm have abnormal distal regions of the tail, in which the plasma membrane is broken and the axonemal components splayed, making the ultrastructural aspects clearly observable. We found that axoneme reduction in the distal region of the sperm does not follow a unique pattern as observed in other insects, but occurs by losing accessory tubules or peripheral doublets. The axonemal tubules contain distinct coaxial ring‐like structures that are still observed after axoneme fragmentation and form large clusters of several units.

The growth of the ciliary axonemes mainly depends on the evolutionary conserved intraflagellar transport (IFT) machinery. However, insect spermatocytes are characterized by cilium‐like regions (CLRs) that elongate in the absence of IFT. It is generally believed that the dynamics of these structures relies on the free diffusion of soluble tubulin from the cytoplasm. However, this passive process could allow the elongation of short ciliary axonemes, but it is unclear whether simple diffusion of tubulin molecules can ensure the correct assembly of elongated ciliary structures. To decipher this point we analyzed the assembly of the CLRs held by the primary spermatocytes of Drosophila bifurca. These ciliary structures consist of a very elongated axoneme that grows without IFT and, therefore, could represent a good model in which to evaluate the role played by the free diffusion of soluble tubulin. The observation of wavy microtubules in the axonemal lumen of fully elongated CLRs of D. bifurca may be consistent with the diffusion of tubulin within the axonemal lumen. Progressive consumption of soluble tubulin used for axoneme growth at the apical tip of the CLRs could result in a gradient sufficient to move tubulin from the cytoplasm to the apical end of the forming ciliary structure. When the axoneme reaches its full length, tubulin molecules are not drawn to the tip of the CLRs and accumulate at the base of the axoneme, where its concentration may exceed the threshold need for microtubule polymerization. The presence of γ‐TuRCs at the proximal ends of the supernumerary microtubules could enhance their nucleation.

The relationship between cytoplasmic incompatibility and the obligate intracellular alphaproteobacteria Wolbachia has for a long time been reported. Although the molecular mechanisms responsible for this reproductive alteration are beginning to be understood, the effects of Wolbachia on germ cell structure and dynamics have not yet been fully investigated. We report here that the presence of Wolbachia in infected cysts of elongating spermatids is associated with major structural defects that become more evident in mature sperm. We find mitochondrial defects, an improper axoneme structure, reduced sperm numbers, and individualization failures. The large heterogeneous variety of the ultrastructural defects found in elongating spermatids and mature sperm provide the first cytological evidence for the reduced fertility associated with Wolbachia infection in Drosophila simulans males. The observed abnormalities could be the result of the mechanical stress induced by the high bacteria numbers during the process of spermatid elongation, rather than the result of the released factors affecting the proper morphogenesis of the germ cells. Moreover, high Wolbachia densities in male germ cells may not be appropriate for causing cytoplasmic incompatibility as the bacteria are harmful for spermatid differentiation, leading to abnormal sperm that is unlikely to be functional.

Induced pluripotent stem cell-derived brain organoids enable the developmental complexities of the human brain to be deconstructed. During embryogenesis, optic vesicles (OVs), the eye primordium attached to the forebrain, develop from diencephalon. However, most 3D culturing methods generate either brain or retinal organoids individually. Here we describe a protocol to generate organoids with both forebrain entities, which we call OV-containing brain organoids (OVB organoids). In this protocol, we first induce neural differentiation (days 0–5) and collect neurospheres, which we culture in a neurosphere medium to initiate their patterning and further self-assembly (days 5–10). Then, upon transfer to spinner flasks containing OVB medium (days 10–30), neurospheres develop into forebrain organoids with one or two pigmented dots restricted to one pole, displaying forebrain entities of ventral and dorsal cortical progenitors and preoptic areas. Further long-term culture results in photosensitive OVB organoids constituting complementary cell types of OVs, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections and electrically active neuronal networks. OVB organoids provide a system to help dissect interorgan interactions between the OVs as sensory organs and the brain as a processing unit, and can help model early eye patterning defects, including congenital retinal dystrophy. To conduct the protocol, experience in sterile cell culture and maintenance of human induced pluripotent stem cells is essential; theoretical knowledge of brain development is advantageous. Furthermore, specialized expertise in 3D organoid culture and imaging for the analysis is needed.

In the model organism insect Drosophila melanogaster short cilia assemble on spermatocytes that elaborate into 1.8 mm long flagella during spermatid differentiation. A unique feature of these cilia/flagella is their lack of dependence on intraflagellar transport (IFT) for their assembly. Here we show that in the common butterfly Pieris brassicae the spermatocyte cilia are exceptionally long: about 40 µm compared to less than 1 µm in Drosophila. By transmission electron microscopy we show that P. brassicae spermatocytes display several features not found in melanogaster, including compelling evidence of IFT structures and features of motile cilia. Summary statement: This work shows ultrastructural definition of the exceptionally long cilia that persist on butterfly ( P. brassicae ) spermatocytes, including evidence of intraflagellar transport, during meiotic division.

Drosophila oogenesis requires the subsequent growth of distinct egg chambers each containing a group of sixteen germline cells surrounded by a simple epithelium of follicle cells. The oocyte occupies a posterior position within the germ cells, thus giving a distinct asymmetry to the egg chamber. Although this disposition is critical for the formation of the anterior–posterior axis of the embryo, the interplay between somatic and germ cells during the early stages of oogenesis remains an open question. We uncover by stage 2, when the egg chambers leaved the germarium, some unique spatial interactions between the posterior follicle cells and the oocyte. These interactions are restricted to the surface of the oocyte over the centriole cluster that formed during early oogenesis. Moreover, the posterior follicle cells in front of the oocyte display a convoluted apical membrane with extensive contacts, whereas the other follicle cells have a flat apical surface without obvious surface protrusions. In addition, the germ cells located at the posterior end of the egg chamber have very elongated protrusions that come into contact with each other or with facing follicle cells. These observations point to distinct polarization events during early oogenesis supporting previous molecular data of an inherent asymmetry between the anterior and the posterior regions of the egg chambers.