Marco Gottardo

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 th...

Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of GSCs’ fundamental biology is a significant bottleneck hindering these efforts. Here, we show th...

During embryogenesis, optic vesicles develop from the diencephalon via a multistep process of organogenesis. Using induced pluripotent stem cell (iPSC)-derived human brain organoids, we attempted to simplify the complexities and demonstrate formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day...

During embryogenesis, optic vesicles develop from the diencephalon via a complex process of organogenesis. Using iPSC-derived human brain organoids, we attempted to simplify the complexities and demonstrate the formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day thirty, brain organoids could...

Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. GBM has a poor prognosis, and currently, there are no curative options exist. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of the fundamental b...

Cilia and flagella are conserved eukaryotic organelles essential for cellular signaling and motility. Cilia dysfunctions cause life-threatening ciliopathies, many of which are due to defects in the transition zone (TZ), a complex structure of the ciliary base. Therefore, understanding TZ assembly, which relies on ordered interactions of multiprotei...

During the extended prophase of Drosophila gametogenesis, spermatocytes undergo robust gene transcription and store many transcripts in the cytoplasm in a repressed state, until translational activation of select mRNAs in later steps of spermatogenesis. Here we characterize the Drosophila Doublefault (Dbf) protein as a C2H2-zinc finger protein, pri...

Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophil...

The fruit-fly Drosophila melanogaster harbours different types of ciliary structures: ciliary projections associated with neurons of type I and cilium-like regions (CLRs) found during male gametogenesis. The latter deserve particular attention since they are morphologically similar to vertebrate primary cilia and transform into the sperm axonemes d...

Centrobin homologues identified in different species localize on daughter centrioles. In Drosophila melanogaster sensory neurons, Centrobin (referred to as CNB in Drosophila ) inhibits basal body function. These data open the question of CNB’s role in spermatocytes, where daughter and mother centrioles become basal bodies. In this study, we report...

We demonstrate that a Drosophila Golgi protein, Gorab, is present not only in the trans-Golgi but also in the centriole cartwheel where, complexed to Sas6, it is required for centriole duplication. In addition to centriole defects, flies lacking Gorab are uncoordinated due to defects in sensory cilia, which lose their nine-fold symmetry. We demonst...

In the developing Drosophila eye the centrioles of the differentiating retinal cells are not surrounded by the microtubule nucleating γ-tubulin suggesting that they are unable to organize functional microtubule organizing centers. Consistent with this idea Cnn and Spd-2, involved in γ-tubulin recruitment, and the scaffold protein Plp that plays a r...

Genome wide screens are widely believed to have identified the great majority of genes required for centriole duplication. However, seeking to clarify the partners of the Drosophila cartwheel protein Sas6, we identified Gorab, a known trans-Golgi associated protein that is mutated in the human wrinkly skin disease, gerodermia osteodysplastica. We n...

Building a new organism usually requires the contribution of two differently shaped haploid cells, the male and female gametes, each providing its genetic material to restore diploidy of the new born zygote. The successful execution of this process requires defined sequential steps that must be completed in space and time. Otherwise, development fa...

Parasitic castration is an adaptive strategy where parasites usurp the hosts’ reproductive physiology to complete their life cycle. The alterations in the host traits vary in their magnitude, from subtle changes in the host morpho-physiology and behaviour to the production of complex aberrant phenotypes, which often depend on the host gender. The s...

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a cl...

Cytokinesis requires a tight coordination between actomyosin ring constriction and new membrane addition along the ingressing cleavage furrow. However, the molecular mechanisms underlying vesicle trafficking to the equatorial site and how this process is coupled with the dynamics of the contractile apparatus are poorly defined. Here we provide evid...

Figure S1. Figure S1. The gene omelette encodes the Drosophila orthologue of Rab1

Figure S3 Validation of mouse anti-Rab1 and rabbit anti Rab1 antibodies by Western Blot and immunofluorescence assays.

Figure S2 Localization of Pav protein at the cleavage site depends on Rab1 function

Mutations in Klp10A, a microtubule-depolymerising Kinesin-13, lead to overly long centrioles in Drosophila male germ cells. We demonstrated that the loss of Klp10A modifies the distribution of typical proteins involved in centriole assembly and function. In the absence of Klp10A the distribution of Drosophila pericentrin-like protein (Dplp), Sas-4...

Aschiphasmatinae is a small group of stick insects from the Oriental region whose genital morphology has been rarely described in detail. The subfamily is of particular interest, as phylogenetic studies have shown Aschiphasmatinae to be the sister group to the remaining Euphasmatodea. In this paper, the male and female terminalia are described for...

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-t...

Microtubule depolymerization assay of F375A PN2-3 mutant which is unable to depolymerize microtubule. The movie was recorded on TIRF microscopy (NikonEclipse Ti) within 10 min after adding PN2-3F375A.

Second example of live cell imaging. Centrioles of control cells stably expressing CPAP-GFP were recorded by time-lapse fluorescence microscopy using 40x water objective. Frames were taken every 5 min. Time counter shows hr:min.

Microtubule depolymerization assay of wild type PN2-3 showing its capability in microtubule sequestration. The movie was recorded on TIRF (total internal reflection fluorescence) microscopy (NikonEclipse Ti) within 10 min after adding PN2-3WT.

TIRF-based microtubule polymerization experiment using buffer control. The movie was recorded on TIRF microscopy (NikonEclipse Ti) within 5 min after adding control buffer.

TIRF-based microtubule polymerization experiment of F375A PN2-3EX mutant. The movie was recorded on TIRF microscopy (NikonEclipse Ti) within 5 min after adding PN2-3EXF375A.

TIRF-based microtubule polymerization experiment of EE343RR PN2-3EX mutant. The movie was recorded on TIRF microscopy (NikonEclipse Ti) within 5 min after adding PN2-3EXEE343RR.

Supplementary Figures 1-12, Supplementary Table 1, Supplementary Methods and Supplementary References

Live cell imaging of CPAPEE343RR expressing RPE1 cells showing a rapid rate of centriolar elongation. Centrioles of cells stably expressing CPAPEE343RR-GFP were recorded by time-lapse fluorescence microscopy using 40x water objective. Movie starts with an interphase cell. At the end of the movie, centriole separation is visible. Square highlights C...

TIRF-based microtubule polymerization experiment of wild type PN2-3EX. The movie was recorded on TIRF microscopy (NikonEclipse Ti) within 5 min after adding PN2-3EXWT.

Microtubule depolymerization assay of EE343RR PN2-3 mutant showing their dynamic capability in microtubule sequestration. The movie was recorded on TIRF microscopy (NikonEclipse Ti) within 10 min after adding PN2-3EE343RR.

Live cell imaging of wild type CPAP (CPAPWT)expressing RPE1 cells. Centrioles of control cells stably expressing CPAP-GFP were recorded by time-lapse fluorescence microscopy using 63x oil objective. Square highlights CPAPWT centrioles. Centrioles of cells expressing CPAPWT remain constant over a period of time, which is known to be approximately 50...

Second example of live cell imaging. Centrioles of cells stably expressing CPAPEE343RR-GFP were recorded by time-lapse fluorescence microscopy using 40x water objective. Movie starts with a mitotic cell in which the centrioles from both of the daughter cells are imaged. At the end of the movie, centriole separation is visible. Square highlights CPA...

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-t...

The Uncoordinated (Unc) gene product, a potential ortholog of orofaciodigital syndrome 1 (Ofd1), is involved in the assembly of the ciliary axoneme in Drosophila and it is, therefore, constrained to cell types that have ciliary structures, namely type 1 sensory neurons and male germ cells. We show that evenly spaced Unc-GFP spots are present in the...

Spermatozoa provide an unusual character system, with a limited number of components organized in a single cell. Similar spermatozoa occur in groups widely separated in the phylogenetic tree of Metazoa. Nevertheless, the character system contains phylogenetic information. Hexapoda have acquired spermatophores along with the switch from aquatic to t...

Appendix

Review Process File

This comprehensive review of the structure of sperm in all orders of insects evaluates phylogenetic implications, with the background of a phylogeny based on transcriptomes. Sperm characters strongly support several major branches of the phylogeny of insects-for instance, Cercophora, Dicondylia, and Psocodea-and also different infraordinal groups....

Cytokinesis culminates in the final separation, or abscission, of the two daughter cells at the end of cell division. Abscission relies on an organelle, the midbody, which forms at the intercellular bridge and is composed of various proteins arranged in a precise stereotypic pattern. The molecular mechanisms controlling midbody organization and fun...

A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms of NPCs maintenance remain unclear. Here, we report an unexpected role for the cilium in NPCs maintenance and identify CPAP as a n...

A new stick insect of the genus Orthomeria Kirby, 1904 (Phasmatodea, Aschiphasmatidae) is described from the Philippines. Orthomeria (O.) kangi sp. n. is readily distinguished from all other congeners by the distinctive blood red colouration of the costal region of the hind wings. Major features of the external morphology of adults, eggs, and first...

Intraspecific colour variations

Centrioles are required to assemble centrosomes for cell division and cilia for motility and signalling. New centrioles assemble perpendicularly to pre-existing ones in G1-S and elongate throughout S and G2. Fully elongated daughter centrioles are converted into centrosomes during mitosis to be able to duplicate and organize pericentriolar material...

Male gametogenesis in insects is unusual in that the centrioles do not duplicate during the second meiosis and the differentiating spermatids inherit only one centriole. Here we showed that a distinct procentriole formed next the proximal region of the centriole in early S13 spermatids at the onion stage, confirming previous reports of a proximal c...

Sensory cilia are organelles that convey information to the cell from the extracellular environment. In vertebrates, ciliary dysfunction results in ciliopathies that in humans comprise a wide spectrum of developmental disorders [1-3]. In Drosophila, sensory cilia are found only in the neurons of type I sensory organs, but ciliary dysfunction also h...

We report, here, that two distinct centriole lineages exist in Drosophila: somatic centrioles usually composed by microtubule doublets and germ line centrioles characterized by triplets. Remarkably, the transition from doublets to triplets in the testis occurs within the stem cell niche with the formation of the C-tubule. We demonstrated that the o...

Sperm of the dictyopteran key taxon Cryptocercus punctulatus was examined. It has largely maintained a blattodean groundplan condition, with a three-layered acrosome, an elongate nucleus, a single centriole, a conspicuous centriole adjunct material, two connecting bands (=accessory bodies), and a long functional flagellum with a 9+9+2 axoneme provi...

A microtubule organizing centre (MTOC) has been described in the spermatid of the hemipteran Matsucoccus feytaudi (Coccoidea). This structure, revealed as a fluorescent ring by treatment with γ-tubulin antibody, gives rise to a bundle of microtubules which surrounds the elongated cylindrical nucleus. This microtubule bundle has been considered an a...

Aurora A kinase plays an important role in several aspects of cell division, including centrosome maturation and separation, a crucial step for the correct organization of the bipolar spindle. Although it has long been showed that this kinase accumulates at the centrosome throughout mitosis its precise contribution to centriole biogenesis and struc...

Accumulating evidence underscores the T-cell immune synapse (IS) as a site of intense vesicular trafficking, on which productive signaling and cell activation crucially depend. Although the T-cell antigen receptor (TCR) is known to exploit recycling to accumulate to the IS, the specific pathway that controls this process remains to be elucidated. H...

The sperm ultrastructure and the male and female genital apparatus of Zorotypus shannoni were examined and documented in detail, mainly using transmission electron microscopy micrographs. The findings suggest an evolutionary trend shared with Z. hubbardi and Z. impolitus. The three species are characterized by enlarged mitochondrial derivatives and...

Psylloidea are economically important insects causing serious damage to plants by direct feeding and/or vectoring bacterial pathogens. Results reported here indicate the presence of extracellular bacteria in the spermatheca of egg-laying Trioza alacris females. One phylotype, sharing 99 % identity with the non-phytopathogenic bacterium Erwinia tasm...

Zoraptera are a cryptic and enigmatic group of insects. The species diversity is lower than in almost all other groups of Hexapoda, but may be distinctly higher than presently known. Several new species were described from different regions recently. The systematic placement was discussed controversially since the group was discovered 100 years ago...

The male and female reproductive apparatus of Zorotypus magnicaudelli (Malaysia), Z. huxleyi (Ecuador) and Z. weidneri (Brazil) were examined and documented in detail. The genital apparatus and sperm of the three species show only minor differences. The testes are larger in Z. magnicaudelli. Z. huxleyi lacks the helical appendage in the accessory g...

Disengagement of parent centrioles represents the licensing process to restrict centriole duplication exactly once during the cell cycle. However, we provide compelling evidence that this general rule is overridden in insect gametogenesis, when distinct procentrioles are generated during prophase of the first meiosis while parent centrioles are sti...

The growing interest in Leptoglossus occidentalis, the conifer seed bug pest accidentally introduced into Europe in the 1990s, led us to investigate the female reproductive structures of the hymenopteran platygastrid Gryon pennsylvanicum, which is its candidate antagonist for biological control programs. Our study revealed a genital apparatus with...

A very unusual genital apparatus of a species of the enigmatic insect order Zoraptera is described. It contains a unique configuration of two different intromittent organs, one of them coiled, very narrow, and strongly elongated. Hyper elongated genitalia are known in different groups of insects. Males have to accommodate these unwieldy structures...

Pharmacological inhibition of Drosophila Polo kinase with BI2536 has allowed us to re-examine the requirements for Polo during Drosophila male gametogenesis. BI2536-treated spermatocytes persisted in a pro-metaphase state without dividing and had condensed chromosomes that did not separate. Centrosomes failed to recruit γ-tubulin and centrosomin (C...

SUMMARY Host castration represents a mechanism used by parasites to exploit energy resources from their hosts by interfering with their reproductive development or to extend host lifespan by removing risks associated with reproductive activity. One of the most intriguing groups of parasitic castrators is represented by the insects belonging to the...

External morphological features of adult males are described in the stick insect Hermarchus leytensis from the Philippines, a species belonging to the little-known euphasmatodean lineage Stephanacridini. Mouthparts are characterized by some likely specialized features: (1) a dentate dorsal cutting edge on the mandibles; (2) distinct differences in...

The male and female genital apparatuses of the recently discovered ground louse Zorotypus impolitus were examined using light and electron microscopy. The rounded testes and a large seminal vesicle are connected with a complex of four accessory glands by a long tapering ejaculatory duct. Two accessory glands have the same whitish coloration, wherea...

Primary cilia and flagella are distinct structures with different functions in eukaryotic cells. Despite the fact that they share similar basic organization and architecture, a direct developmental continuity among them has not been reported until now. The primary cilium is a dynamic structure that typically assembles and disassembles during mitoti...

A remarkable external sperm transfer is described for the first time in a species of a group of winged insects (Pterygota), the enigmatic Zoraptera. Mating and sperm transfer of two species of the order were examined in detail, documented, and compared with each other and with patterns described for other species belonging to the order. The behavio...

Gryon pennsylvanicum is a platygastrid hymenopteran that has lately received increasing attention in Europe due to its possible use in biological control of the conifer seed bug pest Leptoglossus occidentalis. Here the male reproductive system and the spermatogenesis of this species, along with those of Gryon muscaeformis, are examined ultrastructu...

Podura aquatica is a springtail of uncertain systematic position. Our study dealing with the ultrastructure of the spermatogenesis and oogenesis of this species is a contribution to a better knowledge of both the reproduction and the systematics of the taxon. In the male, the spermatogenesis proceeds in a similar way to that of other Collembola. Pr...