Precise control of microtubule disassembly in living cells

Microtubules (MTs) are components of the evolutionarily conserved cytoskeleton, which tightly regulates various cellular activities. Our understanding of MTs is largely based on MT-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific MT populations due to their slow effects on the entire pool of MTs in cells. To address this limitation, we have used chemogenetics and optogenetics to disassemble specific MT subtypes by rapid recruitment of engineered MT-cleaving enzymes. Acute MT disassembly swiftly halted vesicular trafficking and lysosome dynamics. We also used this approach to disassemble MTs specifically modified by tyrosination and several MT-based structures including primary cilia, mitotic spindles, and intercellular bridges. These effects were rapidly reversed by inhibiting the activity or MT association of the cleaving enzymes. The disassembly of targeted MTs with spatial and temporal accuracy enables to uncover new insights of how MTs precisely regulate cellular architectures and functions.

Intercellular Transfer of Mitochondria between Senescent Cells through Cytoskeleton-Supported Intercellular Bridges Requires mTOR and CDC42 Signalling

Cellular senescence is a state of irreversible cell proliferation arrest induced by various stressors including telomere attrition, DNA damage, and oncogene induction. While beneficial as an acute response to stress, the accumulation of senescent cells with increasing age is thought to contribute adversely to the development of cancer and a number of other age-related diseases, including neurodegenerative diseases for which there are currently no effective disease-modifying therapies. Non-cell-autonomous effects of senescent cells have been suggested to arise through the SASP, a wide variety of proinflammatory cytokines, chemokines, and exosomes secreted by senescent cells. Here, we report an additional means of cell communication utilised by senescent cells via large numbers of membrane-bound intercellular bridges—or tunnelling nanotubes (TNTs)—containing the cytoskeletal components actin and tubulin, which form direct physical connections between cells. We observe the presence of mitochondria in these TNTs and show organelle transfer through the TNTs to adjacent cells. While transport of individual mitochondria along single TNTs appears by time-lapse studies to be unidirectional, we show by differentially labelled co-culture experiments that organelle transfer through TNTs can occur between different cells of equivalent cell age, but that senescent cells, rather than proliferating cells, appear to be predominant mitochondrial donors. Using small molecule inhibitors, we demonstrate that senescent cell TNTs are dependent on signalling through the mTOR pathway, which we further show is mediated at least in part through the downstream actin-cytoskeleton regulatory factor CDC42. These findings have significant implications for the development of senomodifying therapies, as they highlight the need to account for local direct cell-cell contacts as well as the SASP in order to treat cancer and diseases of ageing in which senescence is a key factor.

Altered germline cyst formation and oogenesis in Tex14 mutant mice

ABSTRACT During oocyte differentiation in mouse fetal ovaries, sister germ cells are connected by intercellular bridges, forming germline cysts. Within the cyst, primary oocytes form via gaining cytoplasm and organelles from sister germ cells through germ cell connectivity. To uncover the role of intercellular bridges in oocyte differentiation, we analyzed mutant female mice lacking testis-expressed 14 (TEX14), a protein involved in intercellular bridge formation and stabilization. In Tex14 homozygous mutant fetal ovaries, germ cells divide to form a reduced number of cysts in which germ cells remained connected via syncytia or fragmented cell membranes, rather than normal intercellular bridges. Compared with wild-type cysts, homozygous mutant cysts fragmented at a higher frequency and produced a greatly reduced number of primary oocytes with precocious cytoplasmic enrichment and enlarged volume. By contrast, Tex14 heterozygous mutant germline cysts were less fragmented and generate primary oocytes at a reduced size. Moreover, enlarged primary oocytes in homozygous mutants were used more efficiently to sustain folliculogenesis than undersized heterozygous mutant primary oocytes. Our observations directly link the nature of fetal germline cysts to oocyte differentiation and development.

Precise levels of the Drosophila adaptor protein Dreadlocks maintain the size and stability of germline ring canals

ABSTRACT Intercellular bridges are essential for fertility in many organisms. The developing fruit fly egg has become the premier model system to study intercellular bridges. During oogenesis, the oocyte is connected to supporting nurse cells by relatively large intercellular bridges, or ring canals. Once formed, the ring canals undergo a 20-fold increase in diameter to support the movement of materials from the nurse cells to the oocyte. Here, we demonstrate a novel role for the conserved SH2/SH3 adaptor protein Dreadlocks (Dock) in regulating ring canal size and structural stability in the germline. Dock localizes at germline ring canals throughout oogenesis. Loss of Dock leads to a significant reduction in ring canal diameter, and overexpression of Dock causes dramatic defects in ring canal structure and nurse cell multinucleation. The SH2 domain of Dock is required for ring canal localization downstream of Src64 (also known as Src64B), and the function of one or more of the SH3 domains is necessary for the strong overexpression phenotype. Genetic interaction and localization studies suggest that Dock promotes WASp-mediated Arp2/3 activation in order to determine ring canal size and regulate growth. This article has an associated First Person interview with the first author of the paper.

Cytokinetic abscission is part of the midblastula transition in early zebrafish embryogenesis

Animal cytokinesis ends with the formation of a thin intercellular membrane bridge that connects the two newly formed sibling cells, which is ultimately resolved by abscission. While mitosis is completed within 15 min, the intercellular bridge can persist for hours, maintaining a physical connection between sibling cells and allowing exchange of cytosolic components. Although cell–cell communication is fundamental for development, the role of intercellular bridges during embryogenesis has not been fully elucidated. In this work, we characterized the spatiotemporal characteristics of the intercellular bridge during early zebrafish development. We found that abscission is delayed during the rapid division cycles that occur in the early embryo, giving rise to the formation of interconnected cell clusters. Abscission was accelerated when the embryo entered the midblastula transition (MBT) phase. Components of the ESCRT machinery, which drives abscission, were enriched at intercellular bridges post-MBT and, interfering with ESCRT function, extended abscission beyond MBT. Hallmark features of MBT, including transcription onset and cell shape modulations, were more similar in interconnected sibling cells compared to other neighboring cells. Collectively, our findings suggest that delayed abscission in the early embryo allows clusters of cells to coordinate their behavior during embryonic development.

Intercellular bridges coordinate the transition from pluripotency to meiosis in mouse fetal oocytes

Meiosis is critical to generating oocytes and ensuring female fertility; however, the mechanisms regulating the switch from mitotic primordial germ cells to meiotic germ cells are poorly understood. Here, we implicate intercellular bridges (ICBs) in this state transition. We used three-dimensional in toto imaging to map meiotic initiation in the mouse fetal ovary and revealed a radial geometry of this transition that precedes the established anterior-posterior wave. Our studies reveal that appropriate timing of meiotic entry across the ovary and coordination of mitotic-meiotic transition within a cyst depend on the ICB component Tex14, which we show is required for functional cytoplasmic sharing. We find that Tex14 mutants more rapidly attenuate the pluripotency transcript Dppa3 upon meiotic initiation, and Dppa3 mutants undergo premature meiosis similar to Tex14. Together, these results lead to a model that ICBs coordinate and buffer the transition from pluripotency to meiosis through dilution of regulatory factors.

Planar cell polarity in the larval epidermis of Drosophila and the role of microtubules

We investigate planar cell polarity (PCP) in the Drosophila larval epidermis. The intricate pattern of denticles depends on only one system of PCP, the Dachsous/Fat system. Dachsous molecules in one cell bind to Fat molecules in a neighbour cell to make intercellular bridges. The disposition and orientation of these Dachsous–Fat bridges allows each cell to compare two neighbours and point its denticles towards the neighbour with the most Dachsous. Measurements of the amount of Dachsous reveal a peak at the back of the anterior compartment of each segment. Localization of Dachs and orientation of ectopic denticles help reveal the polarity of every cell. We discuss whether these findings support our gradient model of Dachsous activity. Several groups have proposed that Dachsous and Fat fix the direction of PCP via oriented microtubules that transport PCP proteins to one side of the cell. We test this proposition in the larval cells and find that most microtubules grow perpendicularly to the axis of PCP. We find no meaningful bias in the polarity of microtubules aligned close to that axis. We also reexamine published data from the pupal abdomen and find no evidence supporting the hypothesis that microtubular orientation draws the arrow of PCP.

Intercellular transfer of mitochondria between senescent cells through cytoskeleton-supported intercellular bridges requires mTOR and Cdc42 signalling

Cellular senescence is a state of irreversible cell proliferation arrest induced by various stressors including telomere attrition, DNA damage and oncogene induction. While beneficial as an acute response to stress, accumulation of senescent cells with increasing age is through to contribute adversely to development of cancer and a number of other age-related diseases, including neurodegenerative diseases for which there are currently no effective disease-modifying therapies. Non-cell autonomous effects of senescent cells have been suggested to arise through the SASP, a wide variety of pro-inflammatory cytokines, chemokines and exosomes secreted by senescent cells. Here, we report an additional means of cell communication utilised by senescent cells via large numbers of membrane-bound intercellular bridges - or tunnelling nanotubes (TNTs) - containing the cytoskeletal components actin and tubulin, and which form direct physical connections between cells. We observe the presence of mitochondria in these TNTs, and show organelle transfer through the TNTs to adjacent cells. While transport of individual mitochondria along single TNTs appears unidirectional, we show by differentially labelled co-culture experiments that organelle transfer through TNTs can occur both between different cells within senescent cell populations, and also between senescent and proliferating cells. Using small molecule inhibitors, we demonstrate that senescent cell TNTs are dependent on signalling through the mTOR pathway, which we further show is mediated at least in part through downstream actin-cytoskeleton regulatory factor Cdc42. These findings have significant implications for development of senomodifying therapies, as they highlight the need to account for local direct cell-cell contacts as well as the SASP in order to treat cancer and diseases of ageing in which senescence is a key factor.

Drosophila sperm development and intercellular cytoplasm sharing through ring canals do not require an intact fusome

ABSTRACTAnimal germ cells communicate directly with each other during gametogenesis through intercellular bridges, often called ring canals (RCs), that form as a consequence of incomplete cytokinesis during cell division. Developing germ cells in Drosophila have an additional specialized organelle connecting the cells called the fusome. Ring canals and the fusome are required for fertility in Drosophila females, but little is known about their roles during spermatogenesis. With live imaging, we directly observe the intercellular movement of GFP and a subset of endogenous proteins through RCs during spermatogenesis, from two-cell diploid spermatogonia to clusters of 64 post-meiotic haploid spermatids, demonstrating that RCs are stable and open to intercellular traffic throughout spermatogenesis. Disruption of the fusome, a large cytoplasmic structure that extends through RCs and is important during oogenesis, had no effect on spermatogenesis or male fertility under normal conditions. Our results reveal that male germline RCs allow the sharing of cytoplasmic information that might play a role in quality control surveillance during sperm development.

Spermatogonial asynchrony in Tex14 mutant mice lacking intercellular bridges

The existence of cytoplasmic passages between germ cells and their potential function in the control of the spermatogenic process has long been an intriguing question. Evidence of the important role of such structures, known as intercellular bridges (ICB), in spermatogenesis has been implicated by the failure of spermatogenesis in testis-expressed gene 14 (Tex14) mutant mice, which lack the ICBs, to progress past the pachytene spermatocyte stage. Using these Tex14 mutants, the present study evaluated, for the first time, the behavior and synchrony of the spermatogonial lineage in the absence of ICBs. Our data suggest that the absence of these cytoplasmic connections between cells affects the expansion of the undifferentiated type A (Aundiff) spermatogonia compartment and their transition to A1, resulting in a significant numerical reduction of differentiating A1 spermatogonia, but did not interfere with cell amplification during subsequent mitotic steps of differentiating spermatogonia from A1 through intermediate (In). However, beginning at the type B spermatogonia, the synchrony of differentiation was impaired as some cells showed delayed differentiation compared to their behavior in a normal seminiferous epithelium cycle. Thus although spermatogonial development is able to proceed, in the absence of ICBs in Tex14−/− mutants, the yield of cells, specific steps of differentiation, the synchrony of the cell kinetics, and the subsequent progression in meiosis are quantitatively lower than normal.