Investigation of Single Boron Acceptors at the Cleaved Si:B (111) Surface

The cleaved and (2 x 1) reconstructed (111) surface of p-type Si is investigated by scanning tunneling microscopy (STM). Single B acceptors are identified due to their characteristic voltage-dependent contrast which is explained by a local energetic shift of the electronic density of states caused by the Coulomb potential of the negatively charged acceptor. In addition, detailed analysis of the STM images shows that apparently one orbital is missing at the B site at sample voltages of 0.4 - 0.6 V, corresponding to the absence of a localized dangling-bond state. Scanning tunneling spectroscopy confirms a strongly altered density of states at the B atom due to the different electronic structure of B compared to Si.Comment: 6 pages, 7 figure

Proteins that carry dual targeting signals can act as tethers between peroxisomes and partner organelles.

Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER-mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication

Single-channel images related to Fig 8E and 8G.

(A) Fluorescence microscopic images of indicated strains expressing mNeonGreen-Pex15 (green), mCherry-PTS (magenta), and Sec63-CFP (blue). (B) Fluorescence microscopic images indicated strains expressing mNeonGreen-Pex15 (green), mCherry-PTS (magenta), and Tim50-CFP (blue). (C) Fluorescence microscopic images of control and indicated mutant cells expressing mNeonGreen-Pex15 (green) and mCherry-PTS (magenta). Scale bar represents 5 μm. (TIF)</p

Localization of RFP-PTS and Ptc5 derivatives in Δ<i>mdm10</i> cells.

(A) Localization of Ptc5-RFP-PTS (magenta, upper panel) and RFP-PTS (magenta, lower panel) in mdm10 mutants expressing a synthetic ER–mitochondria tether (green) [43] and quantification of peroxisome number per cell in strains of the lower panel. (B) Fluorescence microscopic images showing expression of the peroxisomal marker RFP-PTS (magenta) in indicated strains. (C) Fluorescence microscopic images showing expression of Ptc5 derivatives in Δmdm10 cells (magenta). Quantifications show the ratio of peroxisomal versus total Ptc5-RFP-PTS signal. The peroxisomal membrane protein Ant1-YFP (green, B and C) and the mitochondrial protein Tim50-CFP (blue, C) served as marker proteins. Scale bar represents 5 μm. Quantifications are based on n = 3 experiments. Each color represents 1 experiment. Error bars represent standard error of the mean. P-values were calculated using a two-sided unpaired Student’s t test. Underlying data for quantifications can be found in S1 Data. (TIF)</p

Analysis of peroxisomes in ERMES mutants upon expression of a synthetic tether.

Images of strains deleted for mdm10 or mdm34 co-expressing RFP-PTS (A, red) or Pxp2-RFP-PTS (B, red), the peroxisomal membrane protein Ant1-YFP (green), and the mitochondrial protein Tim50 (blue) either in the presence of Tom70-ProteinA-TAPex15 or a control protein. Scale bars represent 5 μm. (TIFF)</p

Synthetic genetic interaction between genes encoding protein involved in sorting and tethering.

(A) Fluorescence microscopic images of indicated strains expressing the mitochondrial marker mt-GFP (green) and the peroxisome marker mCherry-PTS (magenta). Scale bar represents 5 μm. (B) Differential centrifugation of post nuclear supernatants of indicated strains. Fractions were analyzed by SDS-PAGE and immunoblot. I, input; 13k, 13k × g pellet; 100k, 100k × g pellet; S, 100k × g supernatant. Pgk1 is a cytosolic protein, Kar2 an ER resident protein. (C) Growth assays of indicated mutants on indicated media. Shown are serial 10-fold dilutions starting with an OD600 = 1. (TIF)</p

Peroxisome function and proximity to mitochondria is affected in ERMES mutants.

(A) Fluorescence microscopic images of control and Δmdm10 cells co-expressing the peroxisomal marker RFP-PTS (magenta) and the peroxisomal membrane protein Ant1-YFP (green) (left). Correlation of the RFP-PTS signal and the Ant1-YFP signal was quantified using PCC (right). (B) The number of peroxisomes per cell was quantified in indicated strains. (C) Quantification of the ratio of peroxisomes in contact with mitochondria (PxM) to the total peroxisome count (PxT) in indicated strains. (D) Scheme of the synthetic PerMit tether. OMM, outer mitochondrial membrane; Px, peroxisome; TA, tail-anchor. (E) A synthetic tether for peroxisomes and mitochondria (Tom70-ProteinA-TAPex15) can suppress accumulation of small peroxisomes. Fluorescence microscopic images of strains deleted for MDM10 expressing RFP-PTS (red) and the marker proteins Ant1-YFP (green) and Tim50-CFP (blue) in presence of the tether Tom70-ProteinA-TAPex15 or a control protein. (F) Fluorescence microscopic images of Δmdm10 cells expressing Ptc5-RFP-PTS (magenta) and Pex3-GFP (green) together with the tether Tom70-ProteinA-TAPex15 or a control protein. Quantifications show the ratio of peroxisomal versus total Ptc5-RFP-PTS signal. (G) Fluorescence microscopic images of strains deleted for MDM10 expressing Pxp2-RFP-PTS (red) and the marker proteins Ant1-YFP (green) and Tim50-CFP (blue) in presence of the tether Tom70-ProteinA-TAPex15 or a control protein. Arrows indicate Pxp2-RFP-PTS foci located at junctions between mitochondria and peroxisomes in Δmdm34 cells. Quantification of the number of peroxisomes per cell of the indicated strains (right). Scale bars represent 5 μm. Quantifications are based on n = 3 experiments. Each color represents 1 experiment. Error bars represent SEM. P-values were calculated using a two-sided unpaired Student’s t test. For multiple comparisons, P-values were calculated with a one-way ANOVA combined with a Tukey test. Underlying data for quantifications can be found in S1 Data. ERMES, endoplasmic reticulum–mitochondria encounter structure; PCC, Pearson’s correlation coefficient; PTS, peroxisome targeting signal; RFP, red fluorescent protein.</p

Inhibition of peroxisomal protein import via an auxin inducible degron reduces PerMit contacts.

(A and B) Images are from indicated strains expressing Ant1-YFP (green), Tim50-CFP (blue), and Cat2-RFP-HA-PTS (red) photographed at indicated time points after addition of 2 mM indole-3-acetic acid (auxin). Arrows indicate peroxisomes that contact mitochondria. (C) Quantifications of contact in control cells lacking Pex13-AID (shown in B) are based on n = 3 experiments. Each color represents 1 experiment. Error bars represent standard error of the mean. P-values were calculated with a one-way ANOVA combined with a Tukey test. Underlying data for quantifications can be found in S1 Data. (TIFF)</p

Expression of the synthetic PerMit tether in control cells.

Fluorescence microscopic images of cells co-expressing the peroxisomal marker RFP-PTS (red, A) or Pxp2-RFP-PTS (red, B), the peroxisomal membrane protein Ant1-YFP (green), and the mitochondrial protein Tim50-CFP. Scale bars represent 5 μm. (TIFF)</p

Inhibition of peroxisomal protein import via an auxin inducible degron reduces PerMit contacts.

(A) Fluorescence microscopic images of indicated strains expressing the peroxisomal membrane protein Ant1-YFP (green) and RFP-PTS (red) in the absence (-Auxin) or presence (+Auxin; 4 h) of 2 mM indole-3-acetic acid. (B and C) Images are from indicated strains expressing Ant1-YFP (magenta) and Tim50-CFP (green) photographed at indicated time points after addition of 2 mM indole-3-acetic acid (auxin). Arrows indicate peroxisomes that contact mitochondria. (D) Quantifications of contacts in control cells lacking Pex13-AID (shown in C) are based on n = 3 experiments. Each color represents 1 experiment. Error bars represent standard error of the mean. P-values were calculated with a one-way ANOVA combined with a Tukey test. Underlying data for quantifications can be found in S1 Data. (TIF)</p