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The Caenorhabditis elegans pericentriolar material components SPD-2 and SPD-5 are monomeric in the cytoplasm before incorporation into the PCM matrix.

Wueseke O, Bunkenborg J, Hein MY, Zinke A, Viscardi V, Woodruff JB, Oegema K, Mann M, Andersen JS, Hyman AA - Mol. Biol. Cell (2014)

Bottom Line: We show that SPD-2 is monomeric, and neither SPD-2 nor SPD-5 exists in complex with PLK-1.SPD-5 exists mostly as a monomer but also forms complexes with the PP2A-regulatory proteins RSA-1 and RSA-2, which are required for microtubule organization at centrosomes.These results suggest that the interactions between SPD-2, SPD-5, and PLK-1 do not result in formation of cytoplasmic complexes, but instead occur in the context of PCM assembly.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany.

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Fluorescence correlation spectroscopy reveals cytoplasmic interactions in vivo. (A) Schematic overview of the experimental procedure. FCS measurements were conducted in the cytoplasm of single-cell embryos at three randomly chosen cytoplasmic positions away from the centrosomes and nucleus. Intensity fluctuations caused by GFP-tagged proteins diffusing through the focal volume were recorded over time. (B) Diffusion of LAP::RSA-1 is affected by depletion or RSA-2 and SPD-5. Autocorrelation curves are depicted as mean ± SEM as calculated from all embryos for each condition. The number of embryos analyzed is indicated next to each condition. Scale bar, 10 μm; white crosses point to exemplary locations of measurements. (C) Diffusion coefficient plot showing diffusion coefficients of LAP::RSA-1. LAP::RSA-1 diffusion changes from 2.0 ± 0.4 to 3.1 ± 0.4 μm2/s (p < 0.001, n = 19) under spd-5 RNAi and 5.1 ± 0.9 μm2/s (p < 0.001, n = 16) under rsa-2 RNAi. Diffusion coefficients were obtained from fitting the autocorrelation curve of LAP::RSA-1 from each embryo. Asterisks indicate significance with p < 0.001. The results show a clear interaction between LAP::RSA-1 and RSA-2, as well as SPD-5. An interaction with PLK-1 or SPD-2 could not be detected. (D) GFP::SPD-2 diffusion is unaffected by depletion of SPD-5 but slightly decreases under PLK-1 depletion. Representation as in B, showing GFP::SPD-2 autocorrelation curves under various conditions. (E) Diffusion coefficient plot of GFP::SPD-2. GFP::SPD-2 diffuses indistinguishably, with ∼2.5 μm2/s in wild- type and spd-5 RNAi conditions. Its diffusion is significantly reduced to ∼2.2 μm2/s under plk-1 RNAi conditions.
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Figure 3: Fluorescence correlation spectroscopy reveals cytoplasmic interactions in vivo. (A) Schematic overview of the experimental procedure. FCS measurements were conducted in the cytoplasm of single-cell embryos at three randomly chosen cytoplasmic positions away from the centrosomes and nucleus. Intensity fluctuations caused by GFP-tagged proteins diffusing through the focal volume were recorded over time. (B) Diffusion of LAP::RSA-1 is affected by depletion or RSA-2 and SPD-5. Autocorrelation curves are depicted as mean ± SEM as calculated from all embryos for each condition. The number of embryos analyzed is indicated next to each condition. Scale bar, 10 μm; white crosses point to exemplary locations of measurements. (C) Diffusion coefficient plot showing diffusion coefficients of LAP::RSA-1. LAP::RSA-1 diffusion changes from 2.0 ± 0.4 to 3.1 ± 0.4 μm2/s (p < 0.001, n = 19) under spd-5 RNAi and 5.1 ± 0.9 μm2/s (p < 0.001, n = 16) under rsa-2 RNAi. Diffusion coefficients were obtained from fitting the autocorrelation curve of LAP::RSA-1 from each embryo. Asterisks indicate significance with p < 0.001. The results show a clear interaction between LAP::RSA-1 and RSA-2, as well as SPD-5. An interaction with PLK-1 or SPD-2 could not be detected. (D) GFP::SPD-2 diffusion is unaffected by depletion of SPD-5 but slightly decreases under PLK-1 depletion. Representation as in B, showing GFP::SPD-2 autocorrelation curves under various conditions. (E) Diffusion coefficient plot of GFP::SPD-2. GFP::SPD-2 diffuses indistinguishably, with ∼2.5 μm2/s in wild- type and spd-5 RNAi conditions. Its diffusion is significantly reduced to ∼2.2 μm2/s under plk-1 RNAi conditions.

Mentions: To confirm that the protein interactions we observed in cytoplasmic extracts also occur in living embryos, we employed fluorescence correlation spectroscopy (FCS) to assess the diffusion of fluorescently tagged proteins in vivo. Protein–protein interactions involving fluorescently tagged proteins can be deduced by combining FCS with RNA interference (RNAi)–mediated depletion of candidate binding partners because depletion of a binding partner is expected to decrease the hydrodynamic radius of complexes containing the tagged protein, thereby increasing its diffusion (Figure 3A). Such differences in diffusion can be observed as time shifts of the autocorrelation curve of the fluorescent protein or by changes in the diffusion coefficient obtained from fitting the autocorrelation curve with a diffusion model. Changes in diffusion strongly depend on the relative change in hydrodynamic radius of the particle and thus are best tested by observing a small molecule under depletion of its larger interaction partner.


The Caenorhabditis elegans pericentriolar material components SPD-2 and SPD-5 are monomeric in the cytoplasm before incorporation into the PCM matrix.

Wueseke O, Bunkenborg J, Hein MY, Zinke A, Viscardi V, Woodruff JB, Oegema K, Mann M, Andersen JS, Hyman AA - Mol. Biol. Cell (2014)

Fluorescence correlation spectroscopy reveals cytoplasmic interactions in vivo. (A) Schematic overview of the experimental procedure. FCS measurements were conducted in the cytoplasm of single-cell embryos at three randomly chosen cytoplasmic positions away from the centrosomes and nucleus. Intensity fluctuations caused by GFP-tagged proteins diffusing through the focal volume were recorded over time. (B) Diffusion of LAP::RSA-1 is affected by depletion or RSA-2 and SPD-5. Autocorrelation curves are depicted as mean ± SEM as calculated from all embryos for each condition. The number of embryos analyzed is indicated next to each condition. Scale bar, 10 μm; white crosses point to exemplary locations of measurements. (C) Diffusion coefficient plot showing diffusion coefficients of LAP::RSA-1. LAP::RSA-1 diffusion changes from 2.0 ± 0.4 to 3.1 ± 0.4 μm2/s (p < 0.001, n = 19) under spd-5 RNAi and 5.1 ± 0.9 μm2/s (p < 0.001, n = 16) under rsa-2 RNAi. Diffusion coefficients were obtained from fitting the autocorrelation curve of LAP::RSA-1 from each embryo. Asterisks indicate significance with p < 0.001. The results show a clear interaction between LAP::RSA-1 and RSA-2, as well as SPD-5. An interaction with PLK-1 or SPD-2 could not be detected. (D) GFP::SPD-2 diffusion is unaffected by depletion of SPD-5 but slightly decreases under PLK-1 depletion. Representation as in B, showing GFP::SPD-2 autocorrelation curves under various conditions. (E) Diffusion coefficient plot of GFP::SPD-2. GFP::SPD-2 diffuses indistinguishably, with ∼2.5 μm2/s in wild- type and spd-5 RNAi conditions. Its diffusion is significantly reduced to ∼2.2 μm2/s under plk-1 RNAi conditions.
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Related In: Results  -  Collection

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Figure 3: Fluorescence correlation spectroscopy reveals cytoplasmic interactions in vivo. (A) Schematic overview of the experimental procedure. FCS measurements were conducted in the cytoplasm of single-cell embryos at three randomly chosen cytoplasmic positions away from the centrosomes and nucleus. Intensity fluctuations caused by GFP-tagged proteins diffusing through the focal volume were recorded over time. (B) Diffusion of LAP::RSA-1 is affected by depletion or RSA-2 and SPD-5. Autocorrelation curves are depicted as mean ± SEM as calculated from all embryos for each condition. The number of embryos analyzed is indicated next to each condition. Scale bar, 10 μm; white crosses point to exemplary locations of measurements. (C) Diffusion coefficient plot showing diffusion coefficients of LAP::RSA-1. LAP::RSA-1 diffusion changes from 2.0 ± 0.4 to 3.1 ± 0.4 μm2/s (p < 0.001, n = 19) under spd-5 RNAi and 5.1 ± 0.9 μm2/s (p < 0.001, n = 16) under rsa-2 RNAi. Diffusion coefficients were obtained from fitting the autocorrelation curve of LAP::RSA-1 from each embryo. Asterisks indicate significance with p < 0.001. The results show a clear interaction between LAP::RSA-1 and RSA-2, as well as SPD-5. An interaction with PLK-1 or SPD-2 could not be detected. (D) GFP::SPD-2 diffusion is unaffected by depletion of SPD-5 but slightly decreases under PLK-1 depletion. Representation as in B, showing GFP::SPD-2 autocorrelation curves under various conditions. (E) Diffusion coefficient plot of GFP::SPD-2. GFP::SPD-2 diffuses indistinguishably, with ∼2.5 μm2/s in wild- type and spd-5 RNAi conditions. Its diffusion is significantly reduced to ∼2.2 μm2/s under plk-1 RNAi conditions.
Mentions: To confirm that the protein interactions we observed in cytoplasmic extracts also occur in living embryos, we employed fluorescence correlation spectroscopy (FCS) to assess the diffusion of fluorescently tagged proteins in vivo. Protein–protein interactions involving fluorescently tagged proteins can be deduced by combining FCS with RNA interference (RNAi)–mediated depletion of candidate binding partners because depletion of a binding partner is expected to decrease the hydrodynamic radius of complexes containing the tagged protein, thereby increasing its diffusion (Figure 3A). Such differences in diffusion can be observed as time shifts of the autocorrelation curve of the fluorescent protein or by changes in the diffusion coefficient obtained from fitting the autocorrelation curve with a diffusion model. Changes in diffusion strongly depend on the relative change in hydrodynamic radius of the particle and thus are best tested by observing a small molecule under depletion of its larger interaction partner.

Bottom Line: We show that SPD-2 is monomeric, and neither SPD-2 nor SPD-5 exists in complex with PLK-1.SPD-5 exists mostly as a monomer but also forms complexes with the PP2A-regulatory proteins RSA-1 and RSA-2, which are required for microtubule organization at centrosomes.These results suggest that the interactions between SPD-2, SPD-5, and PLK-1 do not result in formation of cytoplasmic complexes, but instead occur in the context of PCM assembly.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany.

Show MeSH
Related in: MedlinePlus