Basal Body Formation Research Articles

TonEBP inhibits ciliogenesis by controlling aurora kinase A and regulating centriolar satellite integrity

BackgroundPrimary cilia on the surface of eukaryotic cells serve as sensory antennas for the reception and transmission in various cell signaling pathways. They are dynamic organelles that rapidly form during differentiation and cell cycle exit. Defects in these organelles cause a group of wide-ranging disorders called ciliopathies. Tonicity-responsive enhancer-binding protein (TonEBP) is a pleiotropic stress protein that mediates various physiological and pathological cellular responses. TonEBP is well-known for its role in adaptation to a hypertonic environment, to which primary cilia have been reported to contribute. Furthermore, TonEBP is involved in a wide variety of other signaling pathways, such as Sonic Hedgehog and WNT signaling, that promote primary ciliogenesis, suggesting a possible regulatory role. However, the functional relationship between TonEBP and primary ciliary formation remains unclear.MethodsTonEBP siRNAs and TonEBP-mCherry plasmids were used to examine their effects on cell ciliation rates, assembly and disassembly processes, and regulators. Serum starvation was used as a condition to induce ciliogenesis.ResultsWe identified a novel pericentriolar localization for TonEBP. The results showed that TonEBP depletion facilitates the formation of primary cilia, whereas its overexpression results in fewer ciliated cells. Moreover, TonEBP controlled the expression and activity of aurora kinase A, a major negative regulator of ciliogenesis. Additionally, TonEBP overexpression inhibited the loss of CP110 from the mother centrioles during the early stages of primary cilia assembly. Finally, TonEBP regulated the localization of PCM1 and AZI1, which are necessary for primary cilia formation.ConclusionsThis study proposes a novel role for TonEBP as a pericentriolar protein that regulates the integrity of centriolar satellite components. This regulation has shown to have a negative effect on ciliogenesis. Investigations into cilium assembly and disassembly processes suggest that TonEBP acts upstream of the aurora kinase A - histone deacetylase 6 signaling pathway and affects basal body formation to control ciliogenesis. Taken together, our data proposes previously uncharacterized regulation of primary cilia assembly by TonEBP.

Phylogenetic distribution and expression pattern analyses identified a divergent basal body assembly protein involved in land plant spermatogenesis.

Land plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is a continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning spermatogenesis remain to be elucidated. We successfully identified candidate genes involved in spermatogenesis, deeply divergent BLD10s, by computational analyses combining multiple methods and omics data. We then examined the functions of BLD10s in the liverwort Marchantia polymorpha and the moss Physcomitrium patens. MpBLD10 and PpBLD10 are required for normal basal body (BB) and flagella formation. Mpbld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, and thus MpBLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. We identified orthologs of MpBLD10 and PpBLD10 in diverse Streptophyta and found that MpBLD10 and PpBLD10 are orthologous to BLD10/CEP135 family proteins, which function in BB assembly. However, BLD10s evolved especially quickly in land plants and MpBLD10 might have acquired additional functions in spermatozoid formation through rapid molecular evolution.

Emi2 enables centriole amplification during multiciliated cell differentiation.

Massive centriole amplification during multiciliated cell (MCC) differentiation is a notable example of organelle biogenesis. This process is thought to be enabled by a derived cell cycle state, but the key cell cycle components required for centriole amplification in MCC progenitors remain poorly defined. Here, we show that emi2 (fbxo43) expression is up-regulated and acts in MCC progenitors after cell cycle exit to transiently inhibit anaphase-promoting complex/cyclosome (APC/C)cdh1 activity. We find that this inhibition is required for the phosphorylation and activation of a key cell cycle kinase, plk1, which acts, in turn, to promote different steps required for centriole amplification and basal body formation, including centriole disengagement, apical migration, and maturation into basal bodies. This emi2-APC/C-plk1 axis is also required to down-regulate gene expression essential for centriole amplification after differentiation is complete. These results identify an emi2-APC/C-plk1 axis that promotes and then terminates centriole assembly and basal body formation during MCC differentiation.

The Transformation of the Centrosome into the Basal Body: Similarities and Dissimilarities between Somatic and Male Germ Cells and Their Relevance for Male Fertility.

The sperm flagellum is essential for the transport of the genetic material toward the oocyte and thus the transmission of the genetic information to the next generation. During the haploid phase of spermatogenesis, i.e., spermiogenesis, a morphological and molecular restructuring of the male germ cell, the round spermatid, takes place that includes the silencing and compaction of the nucleus, the formation of the acrosomal vesicle from the Golgi apparatus, the formation of the sperm tail, and, finally, the shedding of excessive cytoplasm. Sperm tail formation starts in the round spermatid stage when the pair of centrioles moves toward the posterior pole of the nucleus. The sperm tail, eventually, becomes located opposed to the acrosomal vesicle, which develops at the anterior pole of the nucleus. The centriole pair tightly attaches to the nucleus, forming a nuclear membrane indentation. An articular structure is formed around the centriole pair known as the connecting piece, situated in the neck region and linking the sperm head to the tail, also named the head-to-tail coupling apparatus or, in short, HTCA. Finally, the sperm tail grows out from the distal centriole that is now transformed into the basal body of the flagellum. However, a centriole pair is found in nearly all cells of the body. In somatic cells, it accumulates a large mass of proteins, the pericentriolar material (PCM), that together constitute the centrosome, which is the main microtubule-organizing center of the cell, essential not only for the structuring of the cytoskeleton and the overall cellular organization but also for mitotic spindle formation and chromosome segregation. However, in post-mitotic (G1 or G0) cells, the centrosome is transformed into the basal body. In this case, one of the centrioles, which is always the oldest or mother centriole, grows the axoneme of a cilium. Most cells of the body carry a single cilium known as the primary cilium that serves as an antenna sensing the cell’s environment. Besides, specialized cells develop multiple motile cilia differing in substructure from the immotile primary cilia that are essential in moving fluids or cargos over the cellular surface. Impairment of cilia formation causes numerous severe syndromes that are collectively subsumed as ciliopathies. This comparative overview serves to illustrate the molecular mechanisms of basal body formation, their similarities, and dissimilarities, in somatic versus male germ cells, by discussing the involved proteins/genes and their expression, localization, and function. The review, thus, aimed to provide a deeper knowledge of the molecular players that is essential for the expansion of clinical diagnostics and treatment of male fertility disorders.

Higher throughput drug screening for rare respiratory diseases: readthrough therapy in primary ciliary dyskinesia.

BackgroundDevelopment of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies followed by ciliated differentiation at the air–liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that can be expanded from such biopsies.MethodsWe describe an immunofluorescence screening method, enabled by extensive expansion of basal cells from PCD patients and the directed differentiation of these cells into ciliated epithelium in miniaturised 96-well transwell format ALI cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia), in this case caused by a homozygous nonsense mutation in the MCIDAS gene.ResultsInitial analyses of ciliary ultrastructure, beat pattern and beat frequency in the 96-well transwell format ALI cultures indicate that a range of different PCD defects can be retained in these cultures. The screening system in our proof-of-principal investigation allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. We observed restoration of basal body formation but not the generation of cilia in the patient's nasal epithelial cells in vitro.ConclusionOur study provides a platform for higher throughput analyses of airway epithelia that is applicable in a range of settings and suggests novel avenues for drug evaluation and development in PCD caused by nonsense mutations.

Anastral Spindle 3/Rotatin Stabilizes Sol narae and Promotes Cell Survival in Drosophila melanogaster.

Apoptosis and compensatory proliferation, two intertwined cellular processes essential for both development and adult homeostasis, are often initiated by the mis-regulation of centrosomal proteins, damaged DNA, and defects in mitosis. Fly Anastral spindle 3 (Ana3) is a member of the pericentriolar matrix proteins and known as a key component of centriolar cohesion and basal body formation. We report here that ana3m19 is a suppressor of lethality induced by the overexpression of Sol narae (Sona), a metalloprotease in a disintegrin and metalloprotease with thrombospondin motif (ADAMTS) family. ana3m19 has a nonsense mutation that truncates the highly conserved carboxyl terminal region containing multiple Armadillo repeats. Lethality induced by Sona overexpression was completely rescued by knockdown of Ana3, and the small and malformed wing and hinge phenotype induced by the knockdown of Ana3 was also normalized by Sona overexpression, establishing a mutually positive genetic interaction between ana3 and sona. p35 inhibited apoptosis and rescued the small wing and hinge phenotype induced by knockdown of ana3. Furthermore, overexpression of Ana3 increased the survival rate of irradiated flies and reduced the number of dying cells, demonstrating that Ana3 actively promotes cell survival. Knockdown of Ana3 decreased the levels of both intra- and extracellular Sona in wing discs, while overexpression of Ana3 in S2 cells dramatically increased the levels of both cytoplasmic and exosomal Sona due to the stabilization of Sona in the lysosomal degradation pathway. We propose that one of the main functions of Ana3 is to stabilize Sona for cell survival and proliferation.

Tissue specific requirement of Drosophila Rcd4 for centriole duplication and ciliogenesis.

Rcd4 is a poorly characterized Drosophila centriole component whose mammalian counterpart, PPP1R35, is suggested to function in centriole elongation and conversion to centrosomes. Here, we show that rcd4 mutants exhibit fewer centrioles, aberrant mitoses, and reduced basal bodies in sensory organs. Rcd4 interacts with the C-terminal part of Ana3, which loads onto the procentriole during interphase, ahead of Rcd4 and before mitosis. Accordingly, depletion of Ana3 prevents Rcd4 recruitment but not vice versa. We find that neither Ana3 nor Rcd4 participates directly in the mitotic conversion of centrioles to centrosomes, but both are required to load Ana1, which is essential for such conversion. Whereas ana3 mutants are male sterile, reflecting a requirement for Ana3 for centriole development in the male germ line, rcd4 mutants are fertile and have male germ line centrioles of normal length. Thus, Rcd4 is essential in somatic cells but is not absolutely required in spermatogenesis, indicating tissue-specific roles in centriole and basal body formation.

KIF14 controls ciliogenesis via regulation of Aurora A and is important for Hedgehog signaling.

Primary cilia play critical roles in development and disease. Their assembly and disassembly are tightly coupled to cell cycle progression. Here, we present data identifying KIF14 as a regulator of cilia formation and Hedgehog (HH) signaling. We show that RNAi depletion of KIF14 specifically leads to defects in ciliogenesis and basal body (BB) biogenesis, as its absence hampers the efficiency of primary cilium formation and the dynamics of primary cilium elongation, and disrupts the localization of the distal appendage proteins SCLT1 and FBF1 and components of the IFT-B complex. We identify deregulated Aurora A activity as a mechanism contributing to the primary cilium and BB formation defects seen after KIF14 depletion. In addition, we show that primary cilia in KIF14-depleted cells are defective in response to HH pathway activation, independently of the effects of Aurora A. In sum, our data point to KIF14 as a critical node connecting cell cycle machinery, effective ciliogenesis, and HH signaling.

Mapping the expression of the sex determining factor Doublesex1 in Daphnia magna using a knock-in reporter

Sexually dimorphic traits are common and widespread among animals. The expression of the Doublesex-/Mab-3-domain (DM-domain) gene family has been widely studied in model organisms and has been proven to be essential for the development and maintenance of sex-specific traits. However, little is known about the detailed expression patterns in non-model organisms. In the present study, we demonstrated the spatiotemporal expression of the DM-domain gene, doublesex1 (dsx1), in the crustacean Daphnia magna, which parthenogenetically produces males in response to environmental cues. We developed a dsx1 reporter strain to track dsx1 activity in vivo by inserting the mCherry gene into the dsx1 locus using the TALEN-mediated knock-in approach. After confirming dsx1 expression in male-specific traits in juveniles and adults, we performed time-lapse imaging of embryogenesis. Shortly after gastrulation stage, a presumptive primary organiser, named cumulus, first showed male-specific dsx1 expression. This cell mass moved to the posterior growth zone that distributes dsx1-expressing progenitor cells across the body during axial elongation, before embryos start male-specific dsx1 expression in sexually dimorphic structures. The present study demonstrated the sex-specific dsx1 expression in cell populations involved in basal body formation.

Identifying domains of EFHC1 involved in ciliary localization, ciliogenesis, and the regulation of Wnt signaling

EFHC1 encodes a ciliary protein that has been linked to Juvenile Myoclonic Epilepsy. In ectodermal explants, derived from Xenopus laevis embryos, the morpholino-mediated down-regulation of EFHC1b inhibited multiciliated cell formation. In those ciliated cells that did form, axoneme but not basal body formation was inhibited. EFHC1b morphant embryos displayed defects in central nervous system (CNS) and neural crest patterning that were rescued by a EFHC1b-GFP chimera. EFHC1b-GFP localized to ciliary axonemes in epidermal, gastrocoele roof plate, and neural tube cells. In X. laevis there is a link between Wnt signaling and multiciliated cell formation. While down-regulation of EFHC1b led to a ~2-fold increase in the activity of the β-catenin/Wnt-responsive TOPFLASH reporter, EFHC1b-GFP did not inhibit β-catenin activation of TOPFLASH. Wnt8a RNA levels were increased in EFHC1b morphant ectodermal explants and intact embryos, analyzed prior to the on-set of ciliogenesis. Rescue of the EFHC1b MO’s ciliary axonemal phenotypes required the entire protein; in contrast, the EFHC1b morpholino’s Wnt8a, CNS, and neural crest phenotypes were rescued by a truncated form of EFHC1b. The EFHC1b morpholino’s Wnt8a phenotype was also rescued by the injection of RNAs encoding secreted Wnt inhibitors, suggesting that these phenotypes are due to effects on Wnt signaling, rather than the loss of cilia, an observation of potential relevance to understanding EFHC1’s role in human neural development.

Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse.

To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development.

Bacillus subtilis Bactofilins Are Essential for Flagellar Hook- and Filament Assembly and Dynamically Localize into Structures of Less than 100 nm Diameter underneath the Cell Membrane.

Bactofilins are a widely conserved protein family implicated in cell shape maintenance and in bacterial motility. We show that the bactofilins BacE and BacF from Bacillus subtilis are essential for motility. The proteins are required for the establishment of flagellar hook- and filament structures, but apparently not for the formation of basal bodies. Functional YFP fusions to BacE and to BacF localize as discrete assemblies at the B. subtilis cell membrane, and have a diameter of 60 to 70 nm. BacF assemblies are relatively static, and partially colocalize with flagellar basal bodies, while BacE assemblies are fewer per cell than those of BacF and are highly mobile. Tracking of BacE foci showed that the assemblies arrest at a single point for a few hundred milliseconds, showing that a putative interaction with flagellar structures would be transient and fast. When overexpressed or expressed in a heterologous cell system, bactofilins can form filamentous structures, and also form multimers as purified proteins. Our data reveal a propensity for bactofilins to form filaments, however, in B. subtilis cells, bactofilins assemble into defined size assemblies that show a dynamic localization pattern and play a role in flagellar assembly.

Control of initial steps of ciliogenesis by protein kinases

Cilia are microtubule-based organelles present on the surface of most vertebrate cells. The formation of the primary cilia requires the mother centriole, which is the older of the two centrioles, to convert into the ciliary basal body. Although recent research has begun to shed light onto the molecular composition of the cilium, the regulation of ciliogenesis is just beginning to be understood. To get insight on the molecular mechanisms that regulate basal body formation, we performed a high-throughput RNA interference screen to identify protein kinases required for ciliogenesis. Of the novel kinases we identified, we are characterizing the function of the microtubule associated/affinity regulating kinase 4 (MARK4), and tau-tubulin kinase 2 (TTBK2). We show that MARK4 and TTBK2 are both required to initiate axoneme extension and to promote the removal of the inhibitory protein complex composed of CP110/Cep97. Together, our data indicate that cilia formation is a highly regulated process, which requires the concerted action of protein kinases that regulate the transition from the mother centriole into the basal body.

Centrin2 regulates CP110 removal in primary cilium formation.

Primary cilia are antenna-like sensory microtubule structures that extend from basal bodies, plasma membrane-docked mother centrioles. Cellular quiescence potentiates ciliogenesis, but the regulation of basal body formation is not fully understood. We used reverse genetics to test the role of the small calcium-binding protein, centrin2, in ciliogenesis. Primary cilia arise in most cell types but have not been described in lymphocytes. We show here that serum starvation of transformed, cultured B and T cells caused primary ciliogenesis. Efficient ciliogenesis in chicken DT40 B lymphocytes required centrin2. We disrupted CETN2 in human retinal pigmented epithelial cells, and despite having intact centrioles, they were unable to make cilia upon serum starvation, showing abnormal localization of distal appendage proteins and failing to remove the ciliation inhibitor CP110. Knockdown of CP110 rescued ciliation in CETN2-deficient cells. Thus, centrin2 regulates primary ciliogenesis through controlling CP110 levels.

Drosophilapericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies

Pericentrin is a critical centrosomal protein required for organizing pericentriolar material (PCM) in mitosis. Mutations in pericentrin cause the human genetic disorder Majewski/microcephalic osteodysplastic primordial dwarfism type II, making a detailed understanding of its regulation extremely important. Germaine to pericentrin's function in organizing PCM is its ability to localize to the centrosome through the conserved C-terminal PACT domain. Here we use Drosophila pericentrin-like-protein (PLP) to understand how the PACT domain is regulated. We show that the interaction of PLP with calmodulin (CaM) at two highly conserved CaM-binding sites in the PACT domain controls the proper targeting of PLP to the centrosome. Disrupting the PLP-CaM interaction with single point mutations renders PLP inefficient in localizing to centrioles in cultured S2 cells and Drosophila neuroblasts. Although levels of PCM are unaffected, it is highly disorganized. We also demonstrate that basal body formation in the male testes and the production of functional sperm does not rely on the PLP-CaM interaction, whereas production of functional mechanosensory neurons does.

Polo-like kinases

Polo-like kinases

Drosophila chibby is required for basal body formation and ciliogenesis but not for Wg signaling

Centriole-to-basal body conversion, a complex process essential for ciliogenesis, involves the progressive addition of specific proteins to centrioles. CHIBBY (CBY) is a coiled-coil domain protein first described as interacting with β-catenin and involved in Wg-Int (WNT) signaling. We found that, in Drosophila melanogaster, CBY was exclusively expressed in cells that require functional basal bodies, i.e., sensory neurons and male germ cells. CBY was associated with the basal body transition zone (TZ) in these two cell types. Inactivation of cby led to defects in sensory transduction and in spermatogenesis. Loss of CBY resulted in altered ciliary trafficking into neuronal cilia, irregular deposition of proteins on spermatocyte basal bodies, and, consequently, distorted axonemal assembly. Importantly, cby(1/1) flies did not show Wingless signaling defects. Hence, CBY is essential for normal basal body structure and function in Drosophila, potentially through effects on the TZ. The function of CBY in WNT signaling in vertebrates has either been acquired during vertebrate evolution or lost in Drosophila.

Extremes in rapid cellular morphogenesis: post-transcriptional regulation of spermatogenesis in Marsilea vestita

The endosporic male gametophyte of the water fern, Marsilea vestita, provides a unique opportunity to study the mechanisms that control cell fate determination during a burst of rapid development. In this review, we show how the spatial and temporal control of development in this simple gametophyte involves several distinct modes of RNA processing that allow the translation of specific mRNAs at distinct stages during gametogenesis. During the early part of development, nine successive cell division cycles occur in precise planes within a closed volume to produce seven sterile cells and 32 spermatids. There is no cell movement in the gametophyte; so, cell position and size within the spore wall define cell fate. After the division cycles have been completed, the spermatids become sites for the de novo formation of basal bodies, for the assembly of a complex cytoskeleton, for nuclear and cell elongation, and for ciliogenesis. In contrast, the adjacent sterile cells exhibit none of these changes. The spermatids differentiate into multiciliated, corkscrew-shaped gametes that resemble no other cells in the entire plant. Development is controlled post-transcriptionally. The transcripts stored in the microspore are released (unmasked) in the gametophyte at different times during development. At the start of these studies, we identified several key mRNAs that undergo translation at specific stages of gametophyte development. We developed RNA silencing protocols that enabled us to block the translation of these proteins and thereby establish their necessity and sufficiency for the completion of specific stages of gametogenesis. In addition, RNAi enabled us to identify additional proteins that are essential for other phases of development. Since the distributions of mRNAs and the proteins they encode are not identical in the gametophyte, transcript processing is apparently important in allowing translation to occur under strict temporal and spatial control. Transcript polyadenylation occurs in the spermatogenous cells in ways that match the translation of specific mRNAs. We have found that the exon junction complex plays key roles in transcript regulation and modifications that underlie cell specification in the gametophyte. We have recently become interested in the mechanisms that control the unmasking of the stored transcripts and have linked the synthesis and redistribution of spermidine in the gametophyte to the control of mRNA release from storage during early development and later to basal body formation, cytoskeletal assembly, and nuclear and cell elongation in the differentiating spermatids.

Spermidine Is a Morphogenetic Determinant for Cell Fate Specification in the Male Gametophyte of the Water FernMarsilea vestita

Here, we show that the polyamine spermidine plays a key role as a morphogenetic determinant during spermatid development in the water fern Marsilea vestita. Spermidine levels rise first in sterile jacket cells and then increase dramatically in spermatogenous cells as the spermatids mature. RNA interference and drug treatments were employed to deplete spermidine in the gametophyte at different stages of gametogenesis. Development in spermidine-depleted gametophytes was arrested before the completion of the last round of cell divisions. In spermidine-depleted spermatogenous cells, chromatin failed to condense properly, basal body positioning was altered, and the microtubule ribbon was in disarray. When cyclohexylamine, a spermidine synthase (SPDS) inhibitor, was added at the start of spermatid differentiation, the spermatid nuclei remained round, centrin failed to localize into basal bodies, thus blocking basal body formation, and the microtubule ribbon was completely abolished. In untreated gametophytes, spermidine made in the jacket cells moves into the spermatids, where it is involved in the unmasking of stored SPDS mRNAs, leading to substantial spermidine synthesis in the spermatids. We found that treating spores directly with spermidine or other polyamines was sufficient to unmask a variety of stored mRNAs in gametophytes and arrest development. Differences in patterns of transcript distribution after these treatments suggest that specific transcripts reside in different locations in the dry spore; these differences may be linked to the timing of unmasking and translation for that mRNA during development.

Centrioles to basal bodies in the spermiogenesis of Mastotermes darwiniensis (Insecta, Isoptera)

In addition to their role in centrosome organization, the centrioles have another distinct function as basal bodies for the formation of cilia and flagella. Centriole duplication has been reported to require two alternate assembly pathways: template or de novo. Since spermiogenesis in the termite Mastotermes darwiniensis lead to the formation of multiflagellate sperm, this process represents a useful model system in which to follow basal body formation and flagella assembly. We present evidence of a possible de novo pathway for basal body formation in the differentiating germ cell. This cell also contains typical centrosomal proteins, such as centrosomin, pericentrin-like protein, gamma-tubulin, that undergo redistribution as spermatid differentiation proceeds. The spermatid centrioles are long structures formed by nine doublet rather than triplet microtubules provided with short projections extending towards the surrounding cytoplasm and with links between doublets. The sperm basal bodies are aligned in parallel beneath the nucleus. They consist of long regions close to the nucleus showing nine doublets in a cartwheel array devoid of any projections; on the contrary, the short region close to the plasma membrane, where the sperm flagella emerge, is characterized by projections similar to those observed in the centrioles linking the basal body to the plasma membrane. It is hypothesized that this appearance is in connection with the centriole elongation and further with the flagellar axonemal organization. Microtubule doublets of sperm flagellar axonemes are provided with outer dynein arms, while inner arms are rarely visible.