Concerted effort of centrosomal and Golgi-derived microtubules is required for proper Golgi complex assembly but not for maintenance

Assembly of an integral Golgi complex is driven by microtubule (MT)-dependent transport. Conversely, the Golgi itself functions as an unconventional MT-organizing center (MTOC). This raises the question of whether Golgi assembly requires centrosomal MTs or can be self-organized, relying on its own MTOC activity. The computational model presented here predicts that each MT population is capable of gathering Golgi stacks but not of establishing Golgi complex integrity or polarity. In contrast, the concerted effort of two MT populations would assemble an integral, polarized Golgi complex. Indeed, while laser ablation of the centrosome did not alter already-formed Golgi complexes, acentrosomal cells fail to reassemble an integral complex upon nocodazole washout. Moreover, polarity of post-Golgi trafficking was compromised under these conditions, leading to strong deficiency in polarized cell migration. Our data indicate that centrosomal MTs complement Golgi self-organization for proper Golgi assembly and motile-cell polarization.     

Centrosome and Golgi complex during differentiation of hepatocytes in early postnatal development of mice

The structure and functional activity of the centrosome was analyzed in hepatocytes of 5-day old mice, as well as the lengths of Golgi complex cisternae. In the early postnatal development of mice, the liver was represented by two types of hepatocytes: in the first type hepatocytes, the centrosome was active as a microtubule organizing center, while in the second type hepatocytes, it was inactive. It was proposed that during ontogenesis the centrosome is inactivated as a microtubule organizing center and activated as an organizing center of intermediate filaments characteristic for differentiated hepatocytes of adult liver. Morphometry of the Golgi complex has shown that Golgi cisternae in the cell center area of early postnatal hepatocytes were longer than in the adult hepatocytes and comparable to those in G ₁-phase hepatocytes of regenerating liver. The possibility of relations between the differences in the Golgi complex morphology and ontogenetic changes in the functional activity of centrosomes is discussed.     

The Golgi apparatus remains associated with microtubule organizing centers during myogenesis

In vitro myogenesis involves a dramatic reorganization of the microtubular network, characterized principally by the relocalization of microtubule nucleating sites at the surface of the nuclei in myotubes, in marked contrast with the classical pericentriolar localization observed in myoblasts (Tassin, A. M., B. Maro, and M. Bornens, 1985, J. Cell Biol., 100:35-46). Since a spatial relationship between the Golgi apparatus and the centrosome is observed in most animal cells, we have decided to follow the fate of the Golgi apparatus during myogenesis by an immunocytochemical approach, using wheat germ agglutinin and an affinity-purified anti-galactosyltransferase. We show that Golgi apparatus in myotubes displays a perinuclear distribution which is strikingly different from the polarized juxtanuclear organization observed in myoblasts. As a result, the Golgi apparatus in myotubes is situated close to the microtubule organizing center (MTOC), the cis-side being situated at a fixed distance from the nuclear envelope, a situation which suggests the existence of a structural association between the Golgi apparatus and the nuclear periphery. This is supported by experiments of microtubule depolymerization by nocodazole, in which a minimal effect was observed on Golgi apparatus localization in myotubes in contrast with the dramatic scattering observed in myoblasts. In both cell types, electron microscopy reveals that microtubule disruption generates individual dictyosomes; this suggests that the connecting structures between dictyosomes are principally affected. This structural dependency of the Golgi apparatus upon microtubules is not apparently accompanied by a reverse dependency of MTOC structure or function upon Golgi apparatus activity. Golgi apparatus modification by monensin, as effective in myotubes as in myoblasts, is without apparent effect on MTOC localization or activity and on microtubule stability. The main result of our study is to show that in a cell type where the MTOC is dissociated from centrioles and where antero-posterior polarity has disappeared, the association between the Golgi apparatus and the MTOC is maintained. The significance of such a tight association is discussed.     

A monoclonal antibody to animal centrosomes recognizes components of the basal-body root complex inChlamydomonas

Summary The mammalian centrosome monoclonal antibody MPM-13 recognized component(s) of the well defined MTOC basal-body root complex in the green plantChlamydomonas. The antibody reaction coincided in location with the basal-body root complex and the cruciate nature of the staining pattern corresponded to the configuration of the root microtubules. During mitosis the behaviour of MPM-13 stained material mirrored the duplication, separation and migration to the spindle poles of the basal body-root complex. It is suggested that conserved MTOC components were recognized and that these may have retained a similar, perhaps universal, function in microtubule organization.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidine-2-phenylindole dihydrochloride - mt mating type - MT microtubule - MTOC microtubule organizing centre - PFA paraformaldehyde - PBS phosphate buffered saline     

A role for a novel centrosome cycle in asymmetric cell division

Tissue stem cells play a key role in tissue maintenance. Drosophila melanogaster central brain neuroblasts are excellent models for stem cell asymmetric division. Earlier work showed that their mitotic spindle orientation is established before spindle formation. We investigated the mechanism by which this occurs, revealing a novel centrosome cycle. In interphase, the two centrioles separate, but only one is active, retaining pericentriolar material and forming a "dominant centrosome." This centrosome acts as a microtubule organizing center (MTOC) and remains stationary, forming one pole of the future spindle. The second centriole is inactive and moves to the opposite side of the cell before being activated as a centrosome/MTOC. This is accompanied by asymmetric localization of Polo kinase, a key centrosome regulator. Disruption of centrosomes disrupts the high fidelity of asymmetric division. We propose a two-step mechanism to ensure faithful spindle positioning: the novel centrosome cycle produces a single interphase MTOC, coarsely aligning the spindle, and spindle-cortex interactions refine this alignment.     

The AE2 anion exchanger is necessary for the structural integrity of the Golgi apparatus in mammalian cells

The structural integrity of the Golgi apparatus is known to be dependent on multiple factors, including the organizational status of microtubules, actin and the ankyrin/spectrin-based Golgi membrane skeleton, as well as vesicular trafficking and pH homeostasis. In this respect, our recently identified Golgi-associated anion exchanger, AE2, may also be of importance, since it potentially acts as a Golgi pH regulator and as a novel membrane anchor for the spectrin-based Golgi membrane skeleton. Here, we show that inhibition (>75%) of AE2 expression by antisense oligonucleotides in COS-7 cells results in the fragmentation of the juxtanuclear Golgi apparatus and in structural disorganization of the Golgi stacks, the cisternae becoming generally shorter, distorted, vesiculated and/or swollen. These structural changes occurred without apparent dissociation of the Golgi membrane skeletal protein Ankyrin(195), but were accompanied by the disappearance of the well-focused microtubule-organizing center (MTOC), suggesting the involvement of microtubule reorganization. Similar changes in Golgi structure and assembly of the MTOC were also observed upon transient overexpression of the EGFP-AE2 fusion protein. These data implicate a clear structural role for the AE2 protein in the Golgi and in its cytological positioning around the MTOC.     

CDC25B is involved in the centrosomal microtubule nucleation in two‐cell stage mouse embryos

CDC25B has been demonstrated to activate the complex of CDK1/Cyclin B and trigger mitosis. We have recently demonstrated that p‐CDC25B‐Ser351 is located at the centrosomes of mouse oocytes and contributes to the release of mouse oocytes from prophase I arrest. But much less is known about CDC25B function at the centrosome in two‐cell stage mouse embryos. Here we investigate the effect of CDC25B regulating the microtubules nucleation. Microinjection of anti‐CDC25B antibody caused aberrant microtubule nucleation. In addition, embryos injected with anti‐CDC25B antibody showed the marked absence of microtubule repolymerization and Nek2 foci after nocodazole washout. CDC25B overexpression caused microtubule‐organizing center (MTOC) overduplication. Moreover, overexpression of CDC25B–?65 mutant resulted in the loss of CDC25B localization in the perinuclear region and made CDC25B less efficient in inducing mitosis. We additionally identified that CDC25B is responsible for the pericentrin localization to the MTOC. Our data suggest an important role of CDC25B for microtubule nucleation and organization. N‐terminal of CDC25B is required for regulating the microtubule dynamics and mitotic function.     

Identification of Microtubule-Organizing Centers in Interphase Melanophores of Xenopus Laevis Larvae In Vivo

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immuno-stained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.     

Strongylocentrotus drobachiensis oocytes maintain a microtubule organizing center throughout oogenesis: implications for the establishment of egg polarity in sea urchins

Although it has been known for over a century that sea urchin eggs are polarized cells, very little is known about the mechanism responsible for establishing and maintaining polarity. Our previous studies of microtubule organization during sea urchin oogenesis described a cortical microtubule-organizing center (MTOC) present during germinal vesicle (GV) migration in large oocytes. This MTOC was localized within the future animal pole of the mature egg. In this study we have used electron microscopy and immunocytochemistry to characterize the structure of this MTOC and have established that this organelle appears prior to GV migration. We show that the cortical MTOC contains all the components of a centrosome, including a pair of centrioles. Although a centrosome proper was not found in small oocytes, the centriole pair in these cells was always found in association with a striated rootlet, a structural remnant of the flagellar apparatus present in precursor germinal cells (PGCs). The centrioles/striated rootlet complex was asymmetrically localized to the side of the oocyte closest to the gonadal wall. These data are consistent with the previously proposed hypothesis that in echinoderms the polarity of the PGCs in the germinal epithelium influences the final polarity of the mature egg.     

Chlamydia trachomatis infection prevents front–rear polarity of migrating HeLa cells

Chlamydiae are obligate intracellular bacterial pathogens that cause trachoma, sexually transmitted diseases and respiratory infections in humans. Fragmentation of the host cell Golgi apparatus (GA) is essential for chlamydial development, whereas the consequences for host cell functions, including cell migration are not well understood. We could show that Chlamydia trachomatis‐infected cells display decelerated migration and fail to repopulate monolayer scratch wounds. Furthermore, infected cells lost the ability to reorient the fragmented GA or the microtubule organization centre (MTOC) after a migratory stimulus. Silencing of golgin‐84 phenocopied this defect in the absence of the infection. Interestingly, GA stabilization via knockdown of Rab6A and Rab11A improved its reorientation in infected cells and it was fully rescued after inhibition of Golgi fragmentation with WEHD‐fmk. These results show that C. trachomatis infection perturbs host cell migration on multiple levels, including the alignment of GA and MTOC.     

An InCytes from MBC Selection: Murine CENP-F Regulates Centrosomal Microtubule Nucleation and Interacts with Hook2 at the Centrosome

The microtubule (MT) network is essential in a broad spectrum of cellular functions. Many studies have linked CENP-F to MT-based activities as disruption of this protein leads to major changes in MT structure and function. Still, the basis of CENP-F regulation of the MT network remains elusive. Here, our studies reveal a novel and critical localization and role for CENP-F at the centrosome, the major MT organizing center (MTOC) of the cell. Using a yeast two-hybrid screen, we identify Hook2, a linker protein that is essential for regulation of the MT network at the centrosome, as a binding partner of CENP-F. With recently developed immunochemical reagents, we confirm this interaction and reveal the novel localization of CENP-F at the centrosome. Importantly, in this first report of CENP-F^−/− cells, we demonstrate that ablation of CENP-F protein function eliminates MT repolymerization after standard nocodazole treatment. This inhibition of MT regrowth is centrosome specific because MT repolymerization is readily observed from the Golgi in CENP-F^−/− cells. The centrosome-specific function of CENP-F in the regulation of MT growth is confirmed by expression of truncated CENP-F containing only the Hook2-binding domain. Furthermore, analysis of partially reconstituted MTOC asters in cells that escape complete repolymerization block shows that disruption of CENP-F function impacts MT nucleation and anchoring rather than promoting catastrophe. Our study reveals a major new localization and function of CENP-F at the centrosome that is likely to impact a broad array of MT-based actions in the cell.     

Can Nocodazole,an Inhibitor of Microtubule Formation,Be Used to Synchronize Mammalian Cells?

Abstract Nocodazole, a temporary inhibitor of microtubule formation, has been used to partly synchronize Ehrlich ascites tumour cells growing in suspension. the gradual entry of cells into mitosis and into the next cell cycle without division during drug treatment has been studied by flow cytometric determination of mitotic cells, analysing red and green fluorescence after low pH treatment and acridine orange staining. Determination of the mitotic index (MI) by this method has been combined with DNA distribution analysis to measure cell-cycle phase durations in asynchronous populations growing in the presence of the drug. With synchronized cells, it was shown that in the concentration range 0.4–4.0 μg/l, cells could only be arrested in mitosis for about 7 hr and at 0.04 μg/ml, for about 5 hr. After these time intervals, the DNA content in nocodazole-blocked cells was found to be increased, and, in parallel, the ratio of red and green fluorescence was found to have changed, showing entry of cells into a next cell cycle without division (polyploidization). It was therefore only possible to partially synchronize an asynchronous population by nocodazole. However, a presynchronized population, e.g. selected G₁ cells or metabolically blocked G₁/S cells, were readily and without harmful effect resynchronized in M phase by a short treatment (0.4 μg/ml, 3–4 hr) with nocodazole; after removal of the drug, cells divided and progressed in a highly synchronized fashion through the next cell cycle.     

CAMSAP3-dependent microtubule dynamics regulates Golgi assembly in epithelial cells

The Golgi assembly pattern varies among cell types. In fibroblast cells, the Golgi apparatus concentrates around the centrosome that radiates microtubules; whereas in epithelial cells, whose microtubules are mainly noncentrosomal, the Golgi apparatus accumulates around the nucleus independently of centrosome. Little is known about the mechanisms behind such cell type-specific Golgi and microtubule organization. Here, we show that the microtubule minus-end binding protein Nezha/CAMSAP3 (calmodulin-regulated spectrin-associated protein 3) plays a role in translocation of Golgi vesicles in epithelial cells. This function of CAMSAP3 is supported by CG-NAP (centrosome and Golgi localized PKN-associated protein) through their binding. Depletion of either one of these proteins similarly induces fragmentation of Golgi membranes. Furthermore, we find that stathmin-dependent microtubule dynamics is graded along the radial axis of cells with highest activity at the perinuclear region, and inhibition of this gradient disrupts perinuclear distribution of the Golgi apparatus. We propose that the assembly of the Golgi apparatus in epithelial cells is induced by a multi-step process, which includes CAMSAP3-dependent Golgi vesicle clustering and graded microtubule dynamics.     

Microtubule organization during zoosporogenesis inAllomyces macrogynus

Summary The microtubule cytoskeleton and cytoplasmic organization ofAllomyces macrogynus during zoosporogenesis was studied using light and electron microscopy. Indirect immunofluorescence methods revealed that the microtubule cytoskeleton progressed through three distinct stages of cytoplasmic distribution during zoospore development. During the first 10 minutes of zoosporogenesis, nuclei were strictly located in the periphery of the cytoplasm, and their associated centrosomes were positioned immediately adjacent to the plasma membrane. Microtubules emanated from centrosomes into the surrounding cytoplasm. Within 20 to 30 min after the induction of zoosporangial cleavage, nuclei migrated to new positions throughout the sporangial cytoplasm and microtubule arrays were primarily organized at and emanated from nuclear surfaces. During the final stage of zoosporogenesis, nuclear envelope-associated microtubules were not observed. Instead, primary organization of cytoplasmic microtubules returned to centrosomes (i.e., basal bodies) and flagella formation was evident. The MPM-2 antibody, which recognizes phosphorylated epitopes of several proteins associated with microtubule nucleation, stained centrosome regions throughout zoosporogenesis but did not stain nuclear envelopes.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamino-2-phenylindole - dH₂O deionized water - DMSO dimethyl sulfoxide - DS dilute salts solution - G/5 0.1% glucose medium - LN₂ liquid nitrogen - LSCM laser scanning confocal microscopy - MTOC microtubule-organizing center - PBS phosphate buffered saline - PCM pericentriolar matrix - TEM transmission electron microscopy - VELM videoenhanced light microscopy     

Unusual Centrosome Cycle in Dictyostelium: Correlation of Dynamic Behavior and Structural Changes

Centrosome duplication and separation are of central importance for cell division. Here we provide a detailed account of this dynamic process in Dictyostelium. Centrosome behavior was monitored in living cells using a γ-tubulin–green fluorescent protein construct and correlated with morphological changes at the ultrastructural level. All aspects of the duplication and separation process of this centrosome are unusual when compared with, e.g., vertebrate cells. In interphase the Dictyostelium centrosome is a box-shaped structure comprised of three major layers, surrounded by an amorphous corona from which microtubules emerge. Structural duplication takes place during prophase, as opposed to G₁/S in vertebrate cells. The three layers of the box-shaped core structure increase in size. The surrounding corona is lost, an event accompanied by a decrease in signal intensity of γ-tubulin–green fluorescent protein at the centrosome and the breakdown of the interphase microtubule system. At the prophase/prometaphase transition the separation into two mitotic centrosomes takes place via an intriguing lengthwise splitting process where the two outer layers of the prophase centrosome peel away from each other and become the mitotic centrosomes. Spindle microtubules are now nucleated from surfaces that previously were buried inside the interphase centrosome. Finally, at the end of telophase, the mitotic centrosomes fold in such a way that the microtubule-nucleating surface remains on the outside of the organelle. Thus in each cell cycle the centrosome undergoes an apparent inside-out/outside-in reversal of its layered structure.     

Identification of microtubule-organizing centers in interphase melanophores of Xenopus laevis larvae in vivo.

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immunostained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.     

DNA SYNTHESIS AND MITOSIS IN MERISTEMS: REQUIREMENTS FOR RNA AND PROTEIN SYNTHESIS

Following provision of sucrose to starved, stationary phase pea root meristems, G₁ and G₂ cells enter DNA synthesis and mitosis, respectively. Puromycin (450 μg/ml) and cycloheximide (5 μg/ml) completely prevent this initiation of progression through the cell cycle. Actinomycin D (10 μg/ml) has no effect on the initial entry of G₁ and G₂ cells into S and mitosis, although later entry is prevented. The resistance of the cells to actinomycin D is lost slowly with time in medium without sucrose, suggesting that an RNA required for the resumption of proliferative activity is being gradually lost. The effects of the inhibitors on transitional and proliferative phase meristem cells indicate that such dividing cells do indeed have sufficient of the requisite RNA for 8-12 hr progression through the cycle, but that protein synthesis is required continuously. It is suggested that this RNA is the one lost slowly during starvation, allowing starved cells to reinitiate progression through the cycle in the presence of actinomycin D.     

Kinesin is the motor for microtubule-mediated Golgi-to-ER membrane traffic [published errata appear in J Cell Biol 1995 Mar;128(5):following 988 and 1995 May;129(3):893]

The distribution and dynamics of both the ER and Golgi complex in animal cells are known to be dependent on microtubules; in many cell types the ER extends toward the plus ends of microtubules at the cell periphery and the Golgi clusters at the minus ends of microtubules near the centrosome. In this study we provide evidence that the microtubule motor, kinesin, is present on membranes cycling between the ER and Golgi and powers peripherally directed movements of membrane within this system. Immunolocalization of kinesin at both the light and electron microscopy levels in NRK cells using the H1 monoclonal antibody to kinesin heavy chain, revealed kinesin to be associated with all membranes of the ER/Golgi system. At steady-state at 37 degrees C, however, kinesin was most concentrated on peripherally distributed, pre- Golgi structures containing beta COP and vesicular stomatitis virus glycoprotein newly released from the ER. Upon temperature reduction or nocodazole treatment, kinesin's distribution shifted onto the Golgi, while with brefeldin A (BFA)-treatment, kinesin could be found in both Golgi-derived tubules and in the ER. This suggested that kinesin associates with membranes that constitutively cycle between the ER and Golgi. Kinesin's role on these membranes was examined by microinjecting kinesin antibody. Golgi-to-ER but not ER-to-Golgi membrane transport was found to be inhibited by the microinjected anti-kinesin, suggesting kinesin powers the microtubule plus end-directed recycling of membrane to the ER, and remains inactive on pre-Golgi intermediates that move toward the Golgi complex.     

Transformations of the Cell Center during Cell Differentiation

A question was posed as to how the multicomponent and polyfunctional organelle dynamically changes during metazoan ontogenesis. The centrosome structure is gradually formed and its functions are switched on during early embryogenesis, one of which is the cell center formation. During cell differentiation, the condition of the cell center and surrounding structures may be different: first, the cell center is quite distinct; second, the cell center is absent due to redistribution of the microtubule organizing centers; third, the cell center disappears due to reversible or irreversible inactivation of the centrosome and other centers of microtubule organization. The assembly of the Golgi complex does not depend directly to the cell center presence. In some cell types, the Golgi complex is topologically associated with the cell center, while in others it exists as individual dictyosomes despite the cell center presence. In some other cell types, the common Golgi complex is assembled without the cell center, but in the presence of microtubules that are formed by noncentrosome centers of microtubule organization. In still others, degradation of both the cell center and the common Golgi complex takes place in the case of centrosome inactivation.     

STUDIES OF SPERMATOGENESIS IN THE HEPATICAE V. BLEPHAROPLAST DEVELOPMENT IN MARCHANTIA POLYMORPHA

Coaxial centrioles and a microtubule organizing center (MTOC) constitute each centrosome in spermatid mother cells of Marchantia polymorpha. During cell division the centrosome separates at its midregion and the two centrioles undergo a planar rotation that brings them to lie somewhat staggered and nearly parallel with their proximal ends embedded in osmiophilic granular material similar in appearance to that of the MTOC. Microtubules of the multilayered structure (MLS) arise in this material below the posterior centriole and parallel to its long axis. The rotation of centrioles and the initiation of S₁ tubules below the posterior centriole determine polarity of the incipient blepharoplast. Lower MLS strata are formed under the anterior centriole by the compaction of granular, osmiophilic matrix. Formation and growth of S₂ vertical lamellae occur at the left front edge of the MLS in association with MTOC-like matrix localized near the cell membrane. The MLS enlarges to about 0.4 μm wide by 0.6 μm long and is ovoid in outline except for a short distal projection underlying the posterior centriole. Subsequently the lamellae are transformed into homogenous, osmiophilic matrix that contributes directly to the expansion of all MLS strata including microtubules. The stratum of lamellae is interpreted as a planar MTOC subject to morphogenetic control. Each of the four strata grows proximally while the tapering distal projection lengthens beneath the posterior basal body. Dense matrix above the MLS, apparently elaborated by the S₂ layer, is organized into cartwheel and triplet components of the basal bodies’ proximal extensions. Organization of triplet tubules proceeds from proximal to distal toward preexisting triplets. Osmiophilic matrix contributes to the formation of microtubule keels and osmiophilic crests and may serve as a cementing material that stabilizes the spatial relationships of blepharoplast components. After full expansion of the MLS’ lower strata, the S₂ layer is reorganized into lamellae. Flagellar growth in Marchantia is postulated to involve a process whereby subunits or their precursors are elaborated by the MLS, translocated to the distal end of the flagellum and incorporated into the axonemal tubules. When MLS microtubules elongate to form a long, narrow band, the distal half of the S₂ layer is again in the osmiophilic matrix state.