ALLO-1- and IKKE-1-dependent positive feedback mechanism promotes the initiation of paternal mitochondrial autophagy.

Allophagy is responsible for the selective removal of paternally inherited organelles, including mitochondria, in Caenorhabditis elegans embryos, thereby facilitating the maternal inheritance of mitochondrial DNA. We previously identified two key factors in allophagy: an autophagy adaptor allophagy-1 (ALLO-1) and TBK1/IKKε family kinase IKKE-1. However, the precise mechanisms by which ALLO-1 and IKKE-1 regulate local autophagosome formation remain unclear. In this study, we identify two ALLO-1 isoforms with different substrate preferences during allophagy. Live imaging reveals a stepwise mechanism of ALLO-1 localization with rapid cargo recognition, followed by ALLO-1 accumulation around the cargo. In the ikke-1 mutant, the accumulation of ALLO-1, and not the recognition of cargo, is impaired, resulting in the failure of isolation membrane formation. Our results also suggest a feedback mechanism for ALLO-1 accumulation via EPG-7/ATG-11, a worm homolog of FIP200, which is a candidate for IKKE-1-dependent phosphorylation. This feedback mechanism may underlie the ALLO-1-dependent initiation and progression of autophagosome formation around paternal organelles.

Optimization of Fluorescent Labeling for In Vivo Nanoimaging of Sarcomeres in the Mouse Heart.

The present study was conducted to systematically investigate the optimal viral titer as well as the volume of the adenovirus vector (ADV) that expresses α-actinin-AcGFP in the Z-disks of myocytes in the left ventricle (LV) of mice. An injection of 10 µL ADV at viral titers of 2 to 4 × 1011 viral particles per mL (VP/mL) into the LV epicardial surface consistently expressed α-actinin-AcGFP in myocytes in vivo, with the fraction of AcGFP-expressing myocytes at ~10%. Our analysis revealed that SL was ~1.90-2.15 µm upon heart arrest via deep anesthesia. Likewise, we developed a novel fluorescence labeling method of the T-tubular system by treating the LV surface with CellMask Orange (CellMask). We found that the T-tubular distance was ~2.10-2.25 µm, similar to SL, in the healthy heart in vivo. Therefore, the present high-precision visualization method for the Z-disks or the T-tubules is beneficial to unveiling the mechanisms of myocyte contraction in health and disease in vivo.

In vivo cardiac nano-imaging: A new technology for high-precision analyses of sarcomere dynamics in the heart.

The cardiac pump function is a result of a rise in intracellular Ca2+ and the ensuing sarcomeric contractions [i.e., excitation-contraction (EC) coupling] in myocytes in various locations of the heart. In order to elucidate the heart's mechanical properties under various settings, cardiac imaging is widely performed in today's clinical as well as experimental cardiology by using echocardiogram, magnetic resonance imaging and computed tomography. However, because these common techniques detect local myocardial movements at a spatial resolution of ∼100 µm, our knowledge on the sub-cellular mechanisms of the physiology and pathophysiology of the heart in vivo is limited. This is because (1) EC coupling occurs in the µm partition in a myocyte and (2) cardiac sarcomeres generate active force upon a length change of ∼100 nm on a beat-to-beat basis. Recent advances in optical technologies have enabled measurements of intracellular Ca2+ dynamics and sarcomere length displacements at high spatial and temporal resolution in the beating heart of living rodents. Future studies with these technologies are warranted to open a new era in cardiac research.

Kinesin-14 is Important for Chromosome Segregation During Mitosis and Meiosis in the Ciliate Tetrahymena thermophila.

Ciliates such as Tetrahymena thermophila have two distinct nuclei within one cell: the micronucleus that undergoes mitosis and meiosis and the macronucleus that undergoes amitosis, a type of nuclear division that does not involve a bipolar spindle, but still relies on intranuclear microtubules. Ciliates provide an opportunity for the discovery of factors that specifically contribute to chromosome segregation based on a bipolar spindle, by identification of factors that affect the micronuclear but not the macronuclear division. Kinesin-14 is a conserved minus-end directed microtubule motor that cross-links microtubules and contributes to the bipolar spindle sizing and organization. Here, we use homologous DNA recombination to knock out genes that encode kinesin-14 orthologues (KIN141, KIN142) in Tetrahymena. A loss of KIN141 led to severe defects in the chromosome segregation during both mitosis and meiosis but did not affect amitosis. A loss of KIN141 altered the shape of the meiotic spindle in a way consistent with the KIN141's contribution to the organization of the spindle poles. EGFP-tagged KIN141 preferentially accumulated at the spindle poles during the meiotic prophase and metaphase I. Thus, in ciliates, kinesin-14 is important for nuclear divisions that involve a bipolar spindle.

Axonemal dynein light chain-1 locates at the microtubule-binding domain of the γ heavy chain.

The outer arm dynein (OAD) complex is the main propulsive force generator for ciliary/flagellar beating. In Chlamydomonas and Tetrahymena, the OAD complex comprises three heavy chains (α, ß, and γ HCs) and >10 smaller subunits. Dynein light chain-1 (LC1) is an essential component of OAD. It is known to associate with the Chlamydomonas γ head domain, but its precise localization within the γ head and regulatory mechanism of the OAD complex remain unclear. Here Ni-NTA-nanogold labeling electron microscopy localized LC1 to the stalk tip of the γ head. Single-particle analysis detected an additional structure, most likely corresponding to LC1, near the microtubule-binding domain (MTBD), located at the stalk tip. Pull-down assays confirmed that LC1 bound specifically to the γ MTBD region. Together with observations that LC1 decreased the affinity of the γ MTBD for microtubules, we present a new model in which LC1 regulates OAD activity by modulating γ MTBD's affinity for the doublet microtubule.

Dynamic Change of Cellular Localization of Microtubule-Organizing Center During Conjugation of Ciliate Tetrahymena thermophila.

To obtain a comprehensive picture of microtubule dynamics during conjugation, the mode of sexual reproduction in ciliates, we combined indirect immunofluorescence and three-dimensional imaging using confocal laser-scanning microscope to visualize the cellular localization of DNA, microtubules, and γ-tubulin, the main component of the microtubule-organizing center in mating Tetrahymena cells. As the conjugational stages proceeded, the distribution of γ-tubulin changed drastically and microtubules showed dynamic appearance and disappearance during meiosis, nuclear selection, nuclear exchange, and the development of new macronuclei. This study highlights the involvement of cytoskeletal regulation in the modulation of germline nuclear motilities required for ciliate reproduction.

Kinesin-13 regulates the quantity and quality of tubulin inside cilia.

Kinesin-13, an end depolymerizer of cytoplasmic and spindle microtubules, also affects the length of cilia. However, in different models, depletion of kinesin-13 either lengthens or shortens cilia, and therefore the exact function of kinesin-13 in cilia remains unclear. We generated null mutations of all kinesin-13 paralogues in the ciliate Tetrahymena. One of the paralogues, Kin13Ap, localizes to the nuclei and is essential for nuclear divisions. The remaining two paralogues, Kin13Bp and Kin13Cp, localize to the cell body and inside assembling cilia. Loss of both Kin13Bp and Kin13Cp resulted in slow cell multiplication and motility, overgrowth of cell body microtubules, shortening of cilia, and synthetic lethality with either paclitaxel or a deletion of MEC-17/ATAT1, the α-tubulin acetyltransferase. The mutant cilia assembled slowly and contained abnormal tubulin, characterized by altered posttranslational modifications and hypersensitivity to paclitaxel. The mutant cilia beat slowly and axonemes showed reduced velocity of microtubule sliding. Thus kinesin-13 positively regulates the axoneme length, influences the properties of ciliary tubulin, and likely indirectly, through its effects on the axonemal microtubules, affects the ciliary dynein-dependent motility.

Formation and ingression of division furrow can progress under the inhibitory condition of actin polymerization in ciliate Tetrahymena pyriformis.

In eukaryotic cells that multiply by binary fission, the interaction of actin filaments with myosin II in the contractile ring is widely recognized to generate force for membrane ingression into the cleavage furrow; however, the expression of myosin II is restricted in animals, yeast, fungi, and amoeba (collectively, unikonts). No corresponding motor protein capable of forming mini-filaments that could exert sufficient tension to cleave the cell body is found in bikonts, consisting of planta, algae, and most protozoa; however, cells in some bikont lineages multiply by binary fission, as do animal cells. Of these, the ciliate Tetrahymena is known to form an actin ring beneath the division furrow in cytokinesis. Here, we investigated the role of filamentous actin in the cytokinesis of Tetrahymena pyriformis by treating synchronized dividing cells with an actin-inhibiting drug, Latrunculin-A. Video microscopic observation of live cells undergoing cytokinesis was performed, and contrary to expectation, we found that initiation of furrow ingression and its progress are not suppressed under the inhibitory condition of actin polymerization in Tetrahymena cells. We suggest that an actin filament-independent mechanism of binary fission may have been acquired during the evolution in this organism.

ADF/cofilin is not essential but is critically important for actin activities during phagocytosis in Tetrahymena thermophila.

ADF/cofilin is a highly conserved actin-modulating protein. Reorganization of the actin cytoskeleton in vivo through severing and depolymerizing of F-actin by this protein is essential for various cellular events, such as endocytosis, phagocytosis, cytokinesis, and cell migration. We show that in the ciliate Tetrahymena thermophila, the ADF/cofilin homologue Adf73p associates with actin on nascent food vacuoles. Overexpression of Adf73p disrupted the proper localization of actin and inhibited the formation of food vacuoles. In vitro, recombinant Adf73p promoted the depolymerization of filaments made of T. thermophila actin (Act1p). Knockout cells lacking the ADF73 gene are viable but grow extremely slowly and have a severely decreased rate of food vacuole formation. Knockout cells have abnormal aggregates of actin in the cytoplasm. Surprisingly, unlike the case in animals and yeasts, in Tetrahymena, ADF/cofilin is not required for cytokinesis. Thus, the Tetrahymena model shows promise for future studies of the role of ADF/cofilin in vivo.

Amitosis requires γ-tubulin-mediated microtubule assembly in Tetrahymena thermophila.

To reveal the molecular systems involved in the division of a cell and its contents during cell proliferation is one of the major subjects in cell biology. Although cytoskeletal organization during mitosis has been well studied, consensus on the molecular basis of amitosis has not been achieved. Here we adapted an immunofluorescence method and investigated the cellular localization of γ-tubulin and microtubules (MTs) in dividing Tetrahymena. Although the macronucleus (Mac) lacks a bipolar spindle, γ-tubulin and MTs are specifically detected in the dividing Mac and show a marked change in the pattern of localization. First, γ-tubulin and MTs appear in whole Mac, then, γ-tubulin gathers at the center of the Mac where the aster-like structure of MTs forms. On Mac expansion, MTs associated with numerous dots of γ-tubulin are reorganized into longitudinally arranged bundles, suggesting that the mutual sliding of each filament and polymerization of MTs may induce Mac expansion. Moreover, normal Mac expansion and equal segregation of the Mac are severely disturbed when γ-tubulin is shut off. We propose that γ-tubulin-mediated MT assembly is required in Mac amitosis of Tetrahymena.