A role for the centrosome in regulating the rate of neuronal efferocytosis by microglia in vivo
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eLife assessment
This paper is an important contribution to the microglia field and will be of interest to a broad readership in the fields of neurobiology, cell biology and immunology. This work describes fundamental mechanisms of efferocytosis by microglia and uses impressive imaging in zebrafish, in combination with molecular manipulations, to provide compelling data of how centrosome movements synchronize with phagocytic cup formation during microglial efferocytosis of neuronal corpses in vivo.
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Abstract
During brain development, many newborn neurons undergo apoptosis and are engulfed by microglia, the tissue-resident phagocytes of the brain, in a process known as efferocytosis. A hallmark of microglia is their highly branched morphology characterized by the presence of numerous dynamic extensions that these cells use for scanning the brain parenchyma and engulfing unwanted material. The mechanisms driving branch formation and apoptotic cell engulfment in microglia are unclear. By taking a live-imaging approach in zebrafish, we show that while microglia generate multiple microtubule-based branches, they only successfully engulf one apoptotic neuron at a time. Further investigation into the mechanism underlying this sequential engulfment revealed that targeted migration of the centrosome into one branch is predictive of phagosome formation and polarized vesicular trafficking. Moreover, experimentally doubling centrosomal numbers in microglia increases the rate of engulfment and even allows microglia to remove two neurons simultaneously, providing direct supporting evidence for a model where centrosomal migration is a rate-limiting step in branch-mediated efferocytosis. Conversely, light-mediated depolymerization of microtubules causes microglia to lose their typical branched morphology and switch to an alternative mode of engulfment, characterized by directed migration towards target neurons, revealing unexpected plasticity in their phagocytic ability. Finally, building on work focusing on the establishment of the immunological synapse, we identified a conserved signalling pathway underlying centrosomal movement in engulfing microglia.
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eLife assessment
This paper is an important contribution to the microglia field and will be of interest to a broad readership in the fields of neurobiology, cell biology and immunology. This work describes fundamental mechanisms of efferocytosis by microglia and uses impressive imaging in zebrafish, in combination with molecular manipulations, to provide compelling data of how centrosome movements synchronize with phagocytic cup formation during microglial efferocytosis of neuronal corpses in vivo.
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Reviewer #1 (Public Review):
In this paper by Moller et al. the authors investigate the basic cell biological processes by which microglia phagocytose apoptotic neurons. This is an important concept to investigate because it is well known that neural debris is produced and that microglia clear it, but little is known about how molecular mechanisms of how microglia phagocytose that debris. These authors utilize the strength of the zebrafish system to identify the microtubule dynamics are critical during a specific type of microglia phagocytosis. Then, the paper describes the molecular components that contribute to this microtubule-mediated process. The paper is excellent, with exceptional imaging and molecular manipulations that support an overall mechanistic pathway. It will be an important contribution to the microglia field and cited …
Reviewer #1 (Public Review):
In this paper by Moller et al. the authors investigate the basic cell biological processes by which microglia phagocytose apoptotic neurons. This is an important concept to investigate because it is well known that neural debris is produced and that microglia clear it, but little is known about how molecular mechanisms of how microglia phagocytose that debris. These authors utilize the strength of the zebrafish system to identify the microtubule dynamics are critical during a specific type of microglia phagocytosis. Then, the paper describes the molecular components that contribute to this microtubule-mediated process. The paper is excellent, with exceptional imaging and molecular manipulations that support an overall mechanistic pathway. It will be an important contribution to the microglia field and cited in future studies that investigate microglia efferocytosis.
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Reviewer #2 (Public Review):
Möller and colleagues describe a crucial role for the centrosome in tissue resident macrophages in the brain, termed microglia, in limiting the rate of efferocytosis. They undertake a live cell imaging approach in zebrafish to demonstrate that microglia remove dying neurons mainly by extending long cellular branches - a process, which depends on an intact microtubule cytoskeleton. They further establish a relationship between centrosome movement into microglial branches and successful neuronal engulfment. Artificial doubling of centrosome numbers led to enhanced engulfment and simultaneous removal of two cells, while cells with only one centrosome preferentially phagocytose one neuron at a time. Thus, they propose that centrosome polarization is a critical parameter in regulating the rate of microglial …
Reviewer #2 (Public Review):
Möller and colleagues describe a crucial role for the centrosome in tissue resident macrophages in the brain, termed microglia, in limiting the rate of efferocytosis. They undertake a live cell imaging approach in zebrafish to demonstrate that microglia remove dying neurons mainly by extending long cellular branches - a process, which depends on an intact microtubule cytoskeleton. They further establish a relationship between centrosome movement into microglial branches and successful neuronal engulfment. Artificial doubling of centrosome numbers led to enhanced engulfment and simultaneous removal of two cells, while cells with only one centrosome preferentially phagocytose one neuron at a time. Thus, they propose that centrosome polarization is a critical parameter in regulating the rate of microglial efferocytosis.
This is a very interesting manuscript. The conclusions of the work are well supported by the data. The imaging is beautiful.
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Reviewer #3 (Public Review):
In this manuscript Moller et al., perform a lovely characterization of how centrosome movements synchronize with phagocytic cup formation during microglial efferocytosis of neuronal corpses in the larval zebrafish. Using a combination of elegant imaging and reporters tools the authors characterize two modes of phagosome formation, one involving process formation. They describe movements of the actin cytoskeleton, microtubules, and the centrosome in this process, and find that targeted migration of the centrosome into one branch is predictive of 'successful' engulfment, and increasing the number of centrosomes increases microglial engulfment capacity, suggesting it is a rate limiting factor. Finally, they use pharmacology to link this to DAG signaling. Although as the authors note, this process has been …
Reviewer #3 (Public Review):
In this manuscript Moller et al., perform a lovely characterization of how centrosome movements synchronize with phagocytic cup formation during microglial efferocytosis of neuronal corpses in the larval zebrafish. Using a combination of elegant imaging and reporters tools the authors characterize two modes of phagosome formation, one involving process formation. They describe movements of the actin cytoskeleton, microtubules, and the centrosome in this process, and find that targeted migration of the centrosome into one branch is predictive of 'successful' engulfment, and increasing the number of centrosomes increases microglial engulfment capacity, suggesting it is a rate limiting factor. Finally, they use pharmacology to link this to DAG signaling. Although as the authors note, this process has been previously linked to phagocytosis in other cell types and the molecular regulators are well known, the beautiful imaging and the focus on microglia makes this a welcome addition to the field. I have no major concerns.
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