Deconstructing Complexity: Serial Block-Face Electron Microscopic Analysis of the Hippocampal Mossy Fiber Synapse

Wilke, Scott A.; Antonios, Joseph K.; Bushong, Eric A.; Badkoobehi, Ali; Malek, Elmar; Hwang, Minju; Terada, Masako; Ellisman, Mark H.; Ghosh, Anirvan

doi:10.1523/jneurosci.1600-12.2013

Cited by 168 publications

(191 citation statements)

References 39 publications

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“…1851 (Miltenyi Biotec) and subsequently fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 h at 4°C, followed by 1 h in 2% OsO 4 , 1.5% potassium ferrocyanide in 0.15 M cacodylate, and 20 min at room temperature in 1% thyocarbohydrazide to allow additional osmium staining (55). Material was stained en bloc with 2% uranyl acetate and Walton's lead aspartate for maximum membrane contrast as described (55) before embedding in Durcupan Araldite embedding resin. Gametocytes were distinguished from asexual parasites by the presence of an inner membrane complex in the parasites (34).…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial ATP synthase is dispensable in blood-stage Plasmodium berghei rodent malaria but essential in the mosquito phase

Sturm

Mollard

Cozijnsen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

102

115

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Mitochondrial ATP synthase is driven by chemiosmotic oxidation of pyruvate derived from glycolysis. Blood-stage malaria parasites eschew chemiosmosis, instead relying almost solely on glycolysis for their ATP generation, which begs the question of whether mitochondrial ATP synthase is necessary during the blood stage of the parasite life cycle. We knocked out the mitochondrial ATP synthase β subunit gene in the rodent malaria parasite, Plasmodium berghei, ablating the protein that converts ADP to ATP. Disruption of the β subunit gene of the ATP synthase only marginally reduced asexual blood-stage parasite growth but completely blocked mouse-to-mouse transmission via Anopheles stephensi mosquitoes. Parasites lacking the β subunit gene of the ATP synthase generated viable gametes that fuse and form ookinetes but cannot progress beyond this stage. Ookinetes lacking the β subunit gene of the ATP synthase had normal motility but were not viable in the mosquito midgut and never made oocysts or sporozoites, thereby abrogating transmission to naive mice via mosquito bite. We crossed the self-infertile ATP synthase β subunit knockout parasites with a male-deficient, self-infertile strain of P. berghei, which restored fertility and production of oocysts and sporozoites, which demonstrates that mitochondrial ATP synthase is essential for ongoing viability through the female, mitochondrion-carrying line of sexual reproduction in P. berghei malaria. Perturbation of ATP synthase completely blocks transmission to the mosquito vector and could potentially be targeted for disease control. malaria | ATP synthase | ookinetes | mitochondrial endosymbiosis | aerobic respiration

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Section: Discussionmentioning

confidence: 99%

Mitochondrial ATP synthase is dispensable in blood-stage Plasmodium berghei rodent malaria but essential in the mosquito phase

Sturm

Mollard

Cozijnsen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

102

115

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“…For SBEM, the ONHs of two male C57BL/6J mice (aged 3 mo and 9 mo) were processed with procedures modified from Wilke et al (19), essentially as previously described by Nguyen et al (4). Volumes were processed and analyzed using IMOD software (http://bio3d.…”

Section: Methodsmentioning

confidence: 99%

Transcellular degradation of axonal mitochondria

Davis

Kim

Bushong

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

530

422

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It is generally accepted that healthy cells degrade their own mitochondria. Here, we report that retinal ganglion cell axons of WT mice shed mitochondria at the optic nerve head (ONH), and that these mitochondria are internalized and degraded by adjacent astrocytes. EM demonstrates that mitochondria are shed through formation of large protrusions that originate from otherwise healthy axons. A virally introduced tandem fluorophore protein reporter of acidified mitochondria reveals that acidified axonal mitochondria originating from the retinal ganglion cell are associated with lysosomes within columns of astrocytes in the ONH. According to this reporter, a greater proportion of retinal ganglion cell mitochondria are degraded at the ONH than in the ganglion cell soma. Consistently, analyses of degrading DNA reveal extensive mtDNA degradation within the optic nerve astrocytes, some of which comes from retinal ganglion cell axons.Together, these results demonstrate that surprisingly large proportions of retinal ganglion cell axonal mitochondria are normally degraded by the astrocytes of the ONH. This transcellular degradation of mitochondria, or transmitophagy, likely occurs elsewhere in the CNS, because structurally similar accumulations of degrading mitochondria are also found along neurites in superficial layers of the cerebral cortex. Thus, the general assumption that neurons or other cells necessarily degrade their own mitochondria should be reconsidered.mitophagy | phagocytosis T he number, half-life, and morphology of mitochondria vary widely across cell types and are regulated by both intrinsic and extrinsic mechanisms. Mitochondria number is controlled through regulated production (1) and degradation (2), as well as by the regulated fusion and fission of existing mitochondria (3). Damaged mitochondria are removed by mitophagy, a subtype of autophagy that involves the enwrapping of mitochondria in autophagosomes that subsequently fuse with lysosomes to become autophagolysosomes (2). Implicit in the categorization of mitophagy as a subtype of autophagy is the assumption that each cell degrades its own mitochondria.Recently, we described a phenomenon at the optic nerve head (ONH) of WT mice, where evulsions originating from otherwise intact axons are engulfed and degraded by resident phagocytic astrocytes (4). Serial section-based 3D reconstructions obtained through serial block-face scanning electron microscopy (SBEM) revealed that the protrusions on axons and the evulsions near axons were common throughout the ONH in both the glial lamina, where retinal ganglion cell axons are unmyelinated, and in the adjacent myelination transition zone (MTZ). The axonal protrusions and evulsions were, on average, larger than the mean diameter of axons and contained membrane-bound organelles of unknown identity. Results Axonal Protrusions and Evulsions Within the ONH Contain Mitochondria.To determine the identity of the membranous material contained within the axonal evulsions at the ONH, a 3-mo-old WT C57BL/6J mouse was an...

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“…Selected pieces were processed in 2 ml Eppendorf tubes for embedding as previously described (Wilke et al 2013). In brief, after fixation, specimens were washed extensively with Na-cacodylate buffer, pH 7.4, supplemented with 2 mM CaCl 2 , and subjected to double-osmication comprising incubations with 2 % OsO 4 -1.5 % K 4 [Fe (CN) 6 ] in Na-cacodylate/CaCl 2 buffer for 1 h on ice, 1 % thiocarbohydrazide for 20 min at RT and 2 % OsO 4 for 30 min at RT.…”

Section: Sample Preparation For Sbemmentioning

confidence: 99%

How to Bury the Dead: Elimination of Apoptotic Hair Cells from the Hearing Organ of the Mouse

Anttonen

Belevich

Kirjavainen

et al. 2014

JARO

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Hair cell death is a major cause of hearing impairment. Preservation of surface barrier upon hair cell loss is critical to prevent leakage of potassium-rich endolymph into the organ of Corti and to prevent expansion of cellular damage. Understanding of wound healing in this cytoarchitecturally complex organ requires ultrastructural 3D visualization. Powered by the serial block-face scanning electron microscopy, we penetrate into the cell biological mechanisms in the acute response of outer hair cells and glial-like Deiters' cells to ototoxic trauma in vivo. We show that Deiters' cells function as phagocytes. Upon trauma, their phalangeal processes swell and the resulting close cellular contacts allow engulfment of apoptotic cell debris. Apical domains of dying hair cells are eliminated from the inner ear sensory epithelia, an event thought to depend on supporting cells' actomyosin contractile activity. We show that in the case of apoptotic outer hair cells of the organ of Corti, elimination of their apices is preceded by strong cell body shrinkage, emphasizing the role of the dying cell itself in the cleavage. Our data reveal that the resealing of epithelial surface by junctional extensions of Deiters' cells is dynamically reinforced by newly polymerized F-actin belts. By analyzing Cdc42-inactivated Deiters' cells with defects in actin dynamics and surface closure, we show that compromised barrier integrity shifts hair cell death from apoptosis to necrosis and leads to expanded hair cell and nerve fiber damage. Our results have implications concerning therapeutic protective and regenerative interventions, because both interventions should maintain barrier integrity.

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Deconstructing Complexity: Serial Block-Face Electron Microscopic Analysis of the Hippocampal Mossy Fiber Synapse

Cited by 168 publications

References 39 publications

Mitochondrial ATP synthase is dispensable in blood-stage Plasmodium berghei rodent malaria but essential in the mosquito phase

Mitochondrial ATP synthase is dispensable in blood-stage Plasmodium berghei rodent malaria but essential in the mosquito phase

Transcellular degradation of axonal mitochondria

How to Bury the Dead: Elimination of Apoptotic Hair Cells from the Hearing Organ of the Mouse

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