Actin chromobody imaging reveals sub-organellar actin dynamics

Schiavon, Cara R.; Zhang, Tong; Zhao, Bing; Moore, Andrew S.; Wales, Pauline; Andrade, Leonardo R.; Wu, Melissa; Sung, Tsung-Chang; Dayn, Yelena; Feng, Jasmine W.; Quintero, Omar A.; Shadel, Gerald S.; Grosse, Robert; Manor, Uri

doi:10.1038/s41592-020-0926-5

Cited by 39 publications

(52 citation statements)

References 44 publications

(40 reference statements)

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“…Thus, while the effects of INF2 CMT mutations have yet to be studied in neurons, it is reasonable to expect that INF2 CMT mutations will cause an actin-dependent decrease in mitochondrial mobility in axons. Furthermore, since the ER regularly contacts many other organelles, and even appears to drive actin-assembly at ER-organelle contact sites ( Korobova et al, 2013 , 2014 ; Manor et al, 2015 ; Chakrabarti et al, 2018 ; Yang and Svitkina, 2019 ; Schiavon et al, 2020 ), it is quite possible INF2 CMT mutations cause aberrant actin assembly on other organelles, reducing their mobility as well ( Figure 1 ).…”

Section: Cmt Mutations Largely Affect Mitochondrial Mobilitymentioning

confidence: 99%

Impaired Mitochondrial Mobility in Charcot-Marie-Tooth Disease

Schiavon

Shadel

Manor

2021

Front. Cell Dev. Biol.

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Charcot-Marie-Tooth (CMT) disease is a progressive, peripheral neuropathy and the most commonly inherited neurological disorder. Clinical manifestations of CMT mutations are typically limited to peripheral neurons, the longest cells in the body. Currently, mutations in at least 80 different genes are associated with CMT and new mutations are regularly being discovered. A large portion of the proteins mutated in axonal CMT have documented roles in mitochondrial mobility, suggesting that organelle trafficking defects may be a common underlying disease mechanism. This review will focus on the potential role of altered mitochondrial mobility in the pathogenesis of axonal CMT, highlighting the conceptional challenges and potential experimental and therapeutic opportunities presented by this “impaired mobility” model of the disease.

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Section: Cmt Mutations Largely Affect Mitochondrial Mobilitymentioning

confidence: 99%

Impaired Mitochondrial Mobility in Charcot-Marie-Tooth Disease

Schiavon

Shadel

Manor

2021

Front. Cell Dev. Biol.

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“…Two key regulators of this constriction are the actin-nucleating protein Spire1C present on mitochondrial membrane [ 169 ], and the actin polymerizing protein inverted formin2 (INF2) present on ER membrane [ 170 ]. Together, these two proteins mediate the formation of actin filaments around mitochondria [ 171 ], which then interact with Non-Muscle Myosin II (NMII) proteins to exert a force required for constriction [ 167 , 168 ]. Notably, all three NMII homologs (NMIIA, B, and C) have been implicated in mediating mitochondrial fission [ 172 , 173 ].…”

Section: Pathogenic Variants In Proteins Mediating Mitochondrial Dynamics That Cause Peripheral Neuropathymentioning

confidence: 99%

Genetic Neuropathy Due to Impairments in Mitochondrial Dynamics

Sharma

Pfeffer

Shutt

2021

Biology

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Mitochondria are dynamic organelles capable of fusing, dividing, and moving about the cell. These properties are especially important in neurons, which in addition to high energy demand, have unique morphological properties with long axons. Notably, mitochondrial dysfunction causes a variety of neurological disorders including peripheral neuropathy, which is linked to impaired mitochondrial dynamics. Nonetheless, exactly why peripheral neurons are especially sensitive to impaired mitochondrial dynamics remains somewhat enigmatic. Although the prevailing view is that longer peripheral nerves are more sensitive to the loss of mitochondrial motility, this explanation is insufficient. Here, we review pathogenic variants in proteins mediating mitochondrial fusion, fission and transport that cause peripheral neuropathy. In addition to highlighting other dynamic processes that are impacted in peripheral neuropathies, we focus on impaired mitochondrial quality control as a potential unifying theme for why mitochondrial dysfunction and impairments in mitochondrial dynamics in particular cause peripheral neuropathy.

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“…The most recent development is an F-actin-binding nanobody called Actin-Chromobody that claims to have a minimal effect on actin dynamics and no notable effect on cell viability [ 20 ]. However, the binding of the Actin-Chromobody to actin has not yet been characterized at molecular level, leaving the true extent of possible side effects open.…”

Section: Introductionmentioning

confidence: 99%

Structure of the Lifeact–F-actin complex

et al. 2020

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Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as a hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

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Actin chromobody imaging reveals sub-organellar actin dynamics

Cited by 39 publications

References 44 publications

Impaired Mitochondrial Mobility in Charcot-Marie-Tooth Disease

Impaired Mitochondrial Mobility in Charcot-Marie-Tooth Disease

Genetic Neuropathy Due to Impairments in Mitochondrial Dynamics

Structure of the Lifeact–F-actin complex

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