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Drosophila melanogaster: a model system to study centriole elimination and basal body dynamics
No full textThe centrosomes play an essential role in cell and tissue homeostasis, therefore, their structure, function, and number are highly regulated to ensure natural organisms development through the assembly of a multiplicity of protein complexes. Since the organization and integrity of the centrosome depend on its centrioles and pericentriolar material (PCM), understanding the dynamics of these organelles is crucial to decipher the centrosome behaviour. To date, we have a fairly detailed knowledge of the centriole composition and structure and also of the process of duplication and centrosomal maturation. Something is understood about the process of centriole elimination during gametogenesis, but very little is known about how centrioles are eliminated in post-mitotic differentiated cells.
During the development of the Drosophila eye, the centrioles of the differentiating retinal cells do not recruit γ-Tubulin, suggesting that they are unable to organize functional microtubule-organizing centers (MTOCs). Consistent with this hypothesis, this study shows that in Drosophila third instar larvae Cnn and Spd-2, proteins that allow γ-Tubulin recruitment, and DPlp, which is involved in the organization of the pericentriolar material, are not accumulated by centrioles of eye imaginal disc cells. Despite the loss of these essential components of the pericentriolar material, the centrioles are structurally intact and can recruit Asl and ANA-1. Usually, the accumulation of Asl and ANA-1 allows the daughter centrioles to acquire the motherhood condition. Indeed, mother centrioles accumulate properly Plk-4; however, they are not able to duplicate. These findings show that, in this model, the accumulation of Plk-4 is not sufficient to allow centriole duplication. During the progression of pupal development, the centriole number progressively decreases, and structural defects can be observed. These phenotypes suggest that during Drosophila eye development centriole elimination begins with the loss of the structural integrity, rather than with the PCM reduction as occurs in other models. Furthermore, Asl, ANA-1 and Sas-4 are still detectable, indicating that these proteins by themselves are not able to ensure the maintenance of centriole integrity.
Among the essential cellular functions played by centrioles, there is their ability to act as basal bodies to nucleate the axoneme, the supporting structure of cilia and flagella, which perform crucial cellular functions such as signal transduction and cell motility. Given the critical role of centrioles and cilia in cell physiology, mutations in numerous centriolar proteins cause various disorders, including microcephaly, dwarfism and ciliopathies.
Therefore, it is crucial to understand better the mechanisms that regulate the dynamics of centrioles and cilia. In this study, the cilia of Drosophila melanogaster type I sensory neurons have been analysed, to understand the role played by the centriolar proteins Klp10A, Cnb, Gorab and Rcd4 in the dynamics of centrioles and cilia.
In Drosophila wild type sensory neurons, Klp10A (Kinesin-like protein 10A), a member of the kinesin-13 family, is located in the distal part of the transition zone (TZ), just above the UNC–GFP signal. This study shows that mutations in klp10A result in substantial structural defects of sensory neurons such as the over elongation of both centrioles in opposite directions. It has also been observed that the extensions of both centrioles, called proximal and distal basal bodies, show doublets surrounded by electron-dense material and short lateral projections as found in the control TZ. Therefore, the elongated distal regions of the centrioles in klp10A mutants may be equivalent to a TZ. The phenotype observed in klp10A mutant is deeply different from that observed in sensory neurons of mutants for other TZ proteins that are limited to the proximal portion of the TZ. This suggests that Klp10A could be a core component of the ciliary transition zone in Drosophila, specifically associated with the distal region of the TZ where it plays an essential role in centriole elongation and the assembly and maintenance of the ciliary axoneme.
Centrobin (Cnb) is a centrosome-associated protein that localizes specifically at the daughter centrioles. It has been shown that a cnb mutation makes the daughter centrioles, called PBBs in this model, able to act as distal basal bodies (DBBs) to nucleate supernumerary axonemes. This is confirmed by the present study performed on a different cnb mutant strain, suggesting that Cnb acts as a negative regulator of ciliogenesis.
Recently a new centriolar protein required for centriole duplication, called Gorab, has been discovered in Drosophila melanogaster. The cnb-gorab double mutant sensory neurons analysed in this study, show a stronger centriole reduction compared to the single gorab mutant. Consequently, the number of cilia is also severely affected. These findings suggest that in the cnb-gorab mutant, the centriole duplication fails before the basal body formation.
Recent works have identified the human protein called PPP1R35 (Rcd4 in Drosophila - Reduction in Cnn dots 4), that is involved in centriole-to-centrosome conversion (CCC) and centriole elongation. Here we demonstrate that rcd4 mutant sensory neurons show a severe centriole and cilia reduction, accompanied by centriolar fragmentation. This suggests that Rcd4 could be involved in the CCC similarly to its human counterpart.Per via del suo ruolo essenziale nell'omeostasi cellulare e tissutale, la struttura, la funzione e il numero di centrosomi sono altamente regolati per garantire il naturale sviluppo degli organismi, attraverso l'assemblaggio di una molteplicità di complessi proteici. Poiché l'organizzazione e l'integrità del centrosoma dipendono dai centrioli e dal materiale pericentriolare (PCM) che lo compongono, comprendere la dinamica di questi organelli è fondamentale per decifrare il comportamento del centrosoma. Ad oggi, abbiamo una conoscenza abbastanza dettagliata della composizione e della struttura dei centrioli e anche di ciò che riguarda il processo di duplicazione e maturazione centrosomale. Si conosce qualcosa del processo di eliminazione dei centrioli durante la gametogenesi, ma si sa molto poco su come i centrioli vengono eliminati nelle cellule differenziate post-mitotiche.
Durante lo sviluppo dell'occhio di Drosophila, i centrioli delle cellule retiniche in differenziazione non reclutano la γ-Tubulina, suggerendo che non sono in grado di organizzare centri di organizzazione dei microtubuli (MTOC) funzionali. Coerentemente con questa ipotesi, questo studio mostra che Cnn e Spd-2, proteine che consentono il reclutamento di γ-tubulina, e DPlp, che è coinvolta nell'organizzazione del materiale pericentriolare, non vengono accumulati dai centrioli delle cellule del terzo stadio larvale. Nonostante la perdita di questi componenti essenziali del materiale pericentriolare, i centrioli sono strutturalmente intatti e possono reclutare Asl e ANA-1. Di solito, l'accumulo di Asl e ANA-1 consente ai centrioli figli di acquisire la condizione di maternità. Infatti, i centrioli madre accumulano correttamente Plk-4; tuttavia, non sono in grado di duplicare. Questi risultati mostrano che, in questo modello, l'accumulo di Plk-4 non è sufficiente per consentire la duplicazione di centrioli. Durante la progressione dello sviluppo della pupa, il numero di centrioli diminuisce progressivamente, e iniziano a essere osservati difetti strutturali. Questi fenotipi suggeriscono che l'eliminazione dei centrioli inizia con la perdita dell'integrità strutturale, piuttosto che con la riduzione del PCM, come mostrato in altri modelli. Inoltre, Asl, ANA-1 e Sas-4 sono ancora rilevabili, sottolineando che queste proteine da sole non sono in grado di garantire il mantenimento dell'integrità dei centrioli.
Tra le funzioni cellulari essenziali svolte dai centrioli, vi è la loro capacità di agire come basal bodies per nucleare l'assonema, la struttura portante di ciglia e flagelli, che svolgono importanti funzioni cellulari come la trasduzione del segnale e la motilità cellulare. Dato il
ruolo critico dei centrioli e delle ciglia nella fisiologia cellulare, le mutazioni di numerose proteine centriolari causano vari disturbi, tra cui microcefalia, nanismo e ciliopatie. Pertanto, è fondamentale comprendere meglio i meccanismi che regolano la dinamica dei centrioli e delle ciglia. In questo studio sono state analizzate le ciglia dei neuroni sensoriali di tipo I della Drosophila melanogaster, per comprendere il ruolo svolto dalle proteine centriolari Klp10A, Cnb, Gorab e Rcd4 nelle dinamiche di centrioli e ciglia.
Nei neuroni sensoriali di tipo I di Drosophila, Klp10A (Kinesin-like protein 10A), un membro della famiglia delle kinesine 13, si localizza nella parte distale della zona di transizione (TZ), appena sopra il segnale UNC-GFP. Questo studio mostra che la mutazione di klp10A provoca sostanziali difetti strutturali dei neuroni sensoriali, come l'eccessivo allungamento di entrambi i centrioli in direzioni opposte. È stato anche osservato che le estensioni di entrambi i centrioli, chiamati basal bodies prossimale e distale, mostrano doppietti circondati da materiale elettrondenso e brevi sporgenze laterali come si quelle che si trovano nella TZ di controllo. Pertanto, le regioni distali allungate dei centrioli dei mutanti per klp10A, possono essere equivalenti a TZ. Il fenotipo osservato nel mutante klp10A è profondamente diverso da quello osservato nei neuroni sensoriali dei mutanti per altre proteine della TZ che sono limitate alla porzione prossimale. Ciò suggerisce che Klp10A potrebbe essere un componente chiave della zona di transizione ciliare in Drosophila, specificamente associato alla regione distale della TZ dove svolge un ruolo essenziale nell'allungamento dei centrioli e nell'assemblaggio e nell mantenimento dell'assoneema ciliare.
La Centrobina (Cnb) è una proteina centrosomale che si localizza specificamente nei centrioli figli. È stato dimostrato che la mutazione della cnb rende i centrioli figli, chiamati PBB in questo modello, in grado di agire come basal body distali (DBB) per nucleare assonemi soprannumerari. Ciò è confermato da questo studio condotto in un diverso ceppo mutante di cnb che suggerisce che la Cnb agisce come regolatore negativo della ciliogenesi.
In Drosophila melanogaster, è stata scoperta una nuova proteina centriolare essenziale per la duplicazione dei centrioli, Gorab. I neuroni sensoriali del doppio mutante cnb-gorab analizzati in questo studio, mostrano una riduzione più forte dei centrioli rispetto al singolo mutante gorab. Di conseguenza, anche il numero di ciglia è gravemente colpito. Questi risultati suggeriscono che nel mutante cnb-gorab, la duplicazione dei centrioli fallisce prima della formazione del basal body.
Lavori recenti hanno identificato la proteina umana chiamata PPP1R35 (Rcd4 in Drosophila - Reduction in Cnn dots 4), che è coinvolta nella conversione centriolocentrosoma (CCC) e nell’allungamento di centriolo. Le analisi dei neuroni sensoriali mutanti di Rcd4 mostrano una forte riduzione dei centrioli e delle ciglia e anche la frammentazione centriolare. Ciò suggerisce che Rcd4 potrebbe essere coinvolto nella CCC in modo simile alla sua controparte umana
Sas-4 colocalizes with the ciliary rootlets of the drosophila sensory organs
Full text linkThe Drosophila eye displays peculiar sensory organs of unknown function, the mechanosensory bristles, that are intercalated among the adjacent ommatidia. Like the other Drosophila sensory organs, the mechanosensory bristles consist of a bipolar neuron and two tandemly aligned centrioles, the distal of which nucleates the ciliary axoneme and represents the starting point of the ciliary rootlets. We report here that the centriole associated protein Sas-4 colocalizes with the short ciliary rootlets of the mechanosensory bristles and with the elongated rootlets of chordotonal and olfactory neurons. This finding suggests an unexpected cytoplasmic localization of Sas-4 protein and points to a new underscored role for this protein. Moreover, we observed that the sheath cells associated with the sensory neurons also display two tandemly aligned centrioles but lacks ciliary axonemes, suggesting that the dendrites of the sensory neurons are dispensable for the assembly of aligned centrioles and rootlets
Early Drosophila Oogenesis: A Tale of Centriolar Asymmetry
Full text linkAmong the morphological processes that characterize the early stages of Drosophila oogenesis, the dynamic of the centrioles deserves particular attention. We re-examined the architecture and the distribution of the centrioles within the germarium and early stages of the vitellarium. We found that most of the germ cell centrioles diverge from the canonical model and display notable variations in size. Moreover, duplication events were frequently observed within the germarium in the absence of DNA replication. Finally, we report the presence of an unusually long centriole that is first detected in the cystoblast and is always associated with the developing oocyte. This centriole is directly inherited after the asymmetric division of the germline stem cells and persists during the process of oocyte selection, thus already representing a marker for oocyte identification at the beginning of its formation and during the ensuing developmental stages
The Microtubule-Depolymerizing Kinesin-13 Klp10A Is Enriched in the Transition Zone of the Ciliary Structures of Drosophila melanogaster
Full text linkThe precursor of the flagellar axoneme is already present in the primary spermatocytes of Drosophila melanogaster. During spermatogenesis each primary spermatocyte shows a centriole pair that moves to the cell membrane and organizes an axoneme-based structure, the cilium-like region (CLR). The CLRs persist through the meiotic divisions and are inherited by young spermatids. During spermatid differentiation the ciliary caps elongate giving rise to the sperm axoneme. Mutations in Klp10A, a kinesin-13 of Drosophila, results in defects of centriole/CLR organization in spermatocytes and of ciliary cap assembly in elongating spermatids. Reduced Klp10A expression also results in strong structural defects of sensory type I neurons. We show, here, that this protein displays a peculiar localization during male gametogenesis. The Klp10A signal is first detected at the distal ends of the centrioles when they dock to the plasma membrane of young primary spermatocytes. At the onset of the first meiotic prometaphase, when the CLRs reach their full size, Klp10A is enriched in a distinct narrow area at the distal end of the centrioles and persists in elongating spermatids at the base of the ciliary cap. We conclude that Klp10A could be a core component of the ciliary transition zone in Drosophila
The male stem cell niche of Drosophila melanogaster: Interactions between the germline stem cells and the hub
No full textThe Drosophila male stem cell niche is a well characterized structure in which a small cluster of somatic cells send self-renewal signals to neighbouring germ cells. Although the molecular information involved in the stem cell fate have been identified, much less is understand on the mechanisms driving their short-range specific release. Our ultrastructural analysis reveals distinct protrusions of the stem cell plasma membrane that interdigitate with membrane protrusions of the facing hub cells. Some of these protrusions are very elongated and extend into the hub and could correspond to the Mt-Nanotubes. Therefore, the interface between the stem cells and the hub appears more complex than previously reported and the membrane protrusions of the stem cells might represent specialized surface areas involved in the niche-stem cell communication. We also noticed the presence of clathrin-coated vesicles in the germline plasma membrane that might be also involved in delivering information from the hub
A Survey on Information and Communication Technologies for Industry 4.0: State-of-the-Art, Taxonomies, Perspectives, and Challenges
No full textA new industrial revolution is undergoing, based on a number of technological paradigms. The will to foster and guide this phenomenon has been summarized in the expression “Industry 4.0” (I4.0). Initiatives under this term share the vision that many key technologies underlying Cyber-Physical Systems and Big Data Analytics are converging to a new distributed, highly automated, and highly dynamic production network, and that this process needs regulatory and cultural advancements to effectively and timely develop. In this work, we focus on the technological aspect only, highlighting the unprecedented complexity of I4.0 emerging from the scientific literature. While previous works have focused on one or up to four related enablers, we consider ten technological enablers, including besides the most cited Big Data, Internet of Things, and Cloud Computing, also others more rarely considered as Fog and Mobile Computing, Artificial Intelligence, Human-Computer Interaction, Robotics, down to the often overlooked, very recent, or taken for granted Open-Source Software, Blockchain, and the Internet. For each we explore the main characteristics in relation to I4.0 and its interdependencies with other enablers. Finally we provide a detailed analysis of challenges in leveraging each of the enablers in I4.0, evidencing possible roadblocks to be overcome and pointing at possible future directions of research. Our goal is to provide a reference for the experts in some of the technological fields involved, for a reconnaissance of integration and hybridization possibilities with other fields in the endeavor of I4.0, as well as for the laymen, for a high-level grasp of the variety (and often deep history) of the scientific research backing I4.0
Cell-to-Cell Interactions during Early Drosophila Oogenesis: An Ultrastructural Analysis
Full text linkDrosophila 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
A transient microtubule-based structure uncovers a new intrinsic asymmetry between the mother centrioles in the early Drosophila spermatocytes
No full textParent centrioles are characterized in most organisms by individual morphological traits and have distinct asymmetries that provide different functional properties. By contrast, mother and daughter centrioles are morphologically undistinguishable during Drosophila male gametogenesis. Here we report the presence of previously unrecognized microtubule-based structures that extend into the peripheral cytoplasm of the Drosophila polar spermatocytes at the onset of the first meiosis and are positive for the typical centriolar protein Sas-4 and for the kinesin-like protein Klp10A. These structures have a short lifespan and are no longer found in early apolar spermatocytes. Remarkably, each polar spermatocyte holds only one microtubule-based structure that is associated with one of the sister centriole pairs and specifically with the mother centriole. These findings reveal an inherent asymmetry between the parent centrioles at the onset of male meiosis and also uncover unexpected functional properties between the mother centrioles of the same cells
Formazione universitaria degli insegnanti alle TD
No full textIl saggio affronta il problema della formazione degli insegnanti alle tecnologie didattiche, evidenziando come esso richieda un approccio sistemico e modelli formativi che interessino trasversalmente il curricolo
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