Journal: PLoS Biology

Article Title: Distinct Roles for Arp2/3 Regulators in Actin Assembly and Endocytosis

doi: 10.1371/journal.pbio.0060001

Figure Lengend Snippet: (A–G) Null mutants lacking one or both type-I myosin. Cells express Sla2-GFP (A–D) or Abp1-GFP (E–G). (A) MSD plots for wild-type and myo5 Δ patches aligned at the start (left) or end (right) of their lifetimes. The curves on the left are truncated at the median lifetime. (B) Percentage of patches that leave the origin. Mean of values for three segregants is shown. (C) Average time at the origin, from the appearance of a patch until it moved away or disappeared. (D) Phase II movement only. For each patch, data prior to movement were removed. (E) MSD plots for wild-type and mutant patches aligned at the start (left) or end (right) of their lifetimes. On the left, plots are truncated at the median lifetime or 16 s, whichever was less. (F) Percentage of patches that leave the origin, as in (B). (G) Average time at the origin, as in (C). (H–M) Mutants lacking Arp2/3-binding regions of one or both type-I myosins, termed myo3 Δ acidic and myo5 Δ acidic . The panels are similar to those above, with cells expressing Sla2-GFP (H–J) or Abp1-GFP (K–M). (H and K) MSD plots for wild-type or mutant patches aligned at the start (left) or end (right) of their lifetimes. The curves on the left are truncated at the median lifetime. (I and L) Percentage of patches that leave the origin. Mean of values for three segregants is shown. (J and M) Average time at the origin, from the appearance of a patch until it moved away or disappeared. (N–S) Mutants lacking Arp2/3 binding regions of WASp/Las17 and type-I myosins. Cells express Sla2-GFP (N–P) or Abp1-GFP (Q–S). (N and Q) MSD plots for wild-type or mutant patches aligned at the start (left) or end (right) of their lifetimes. The curves on the left are truncated at the median lifetime. (O and P) Percentage of patches that leave the origin. Mean of values for three segregants is shown. (R and S) Average time at the origin, from the appearance of a patch until it moved away or disappeared. Strain numbers and number of patches: (A–C) Wild-type, YJC4787–9; 94, 79, and 90. myo5 Δ, YJC4784–6; 103, 79, and 69. (D) Strains as in (A–C). Numbers of patches: wild-type – 88, 69, 85. myo5 Δ – 75, 40, 36. (E–G) Wild-type, YJC4815–7; 121, 110, and 130. myo3 Δ, YJC4409–11; 111, 115, and 105. myo5 Δ, YJC4813–4; 120 and 99. myo3 Δ myo5 Δ, YJC4770–2; 143, 141, and 126. (H–J) Wild-type, YJC5566–8; 71, 107, and 83. myo3 Δ acidic , YJC5563–5; 71, 75, and 101. myo5 Δ acidic , YJC5554–6; 86, 71, and 62. myo3 Δ acidic myo5 Δ acidic , YJC5557–9; 83, 65, and 83. (K–M) Wild-type, YJC4848 and 50; 116 and 87. myo3 Δ acidic , YJC4843–5; 119, 99, and 106. myo5 Δ acidic , YJC4840–2; 102, 111, and 99. myo3 Δ acidic myo5 Δ acidic , YJC4837–9; 96, 93, and 100. (N–P) Wild-type, YJC5566–8; 71, 107, and 83. las17 Δ acidic , YJC5551–3; 84, 64, and 69. las17 Δ acidic myo3 Δ acidic , YJC5560–2; 89, 71, and 68. las17 Δ acidic myo5 Δ acidic , YJC5548–50; 51, 66, and 57. las17 Δ acidic myo3 Δ acidic myo5 Δ acidic , YJC5545–7; 43, 53, and 54. (Q–S) las17 Δ acidic , YJC5208–10; 95, 76, and 89. las17 Δ acidic myo3 Δ acidic , YJC5438–40; 111, 104, and 109. las17 Δ acidic myo5 Δ acidic , YJC5441–3; 98, 80, and 84. las17 Δ acidic myo3 Δ acidic myo5 Δ acidic , YJC5205–7; 71, 79, and 99.

Article Snippet: Two color images were collected sequentially using a LMM5 Laser Merge Module (Spectral Applied Research), a multipass dichroic mirror, and emission filters in a Lambda 10–3 high-speed filter wheel (Sutter Instruments).

Techniques: Mutagenesis, Binding Assay, Expressing

Journal: PLoS Biology

Article Title: Distinct Roles for Arp2/3 Regulators in Actin Assembly and Endocytosis

doi: 10.1371/journal.pbio.0060001

Figure Lengend Snippet: Cells express Sla2-GFP (A–C) or Abp1-GFP (D–F). ( A and D) MSD plots for wild-type and mutant patches aligned at the start (left) or end (right) of their lifetimes. The curves on the left are truncated at the median lifetime. (B and E) Percentage of patches that leave the origin. Mean of values for three segregants is shown. (C and F) Average time at the origin, from the appearance of a patch until it moved away or disappeared. Strain numbers and numbers of patches: (A–C) Wild-type, YJC5707 - 9; 66, 84, and 69. las17 Δ acidic , YJC5713–5; 100, 95, and 104. pan1 Δ acidic , YJC5710–2; 103, 76, and 74. las17 Δ acidic pan1 Δ acidic , YJC5716–8; 100, 86, and 91. (D–F) Wild-type, YJC5719–22; 99, 100, 114, and 98. las17 Δ acidic , YJC5726, 8 & 9; 95, 88, and 84. pan1 Δ acidic , YJC5723–5; 102, 94, and 139. las17 Δ acidic pan1 Δ acidic , YJC5730–3; 77, 70, 50, and 50. Error bars are ± standard error.

Article Snippet: Two color images were collected sequentially using a LMM5 Laser Merge Module (Spectral Applied Research), a multipass dichroic mirror, and emission filters in a Lambda 10–3 high-speed filter wheel (Sutter Instruments).

Techniques: Mutagenesis

Journal:

Article Title: Differential recruitment of PKC isoforms in HeLa cells during redox stress

doi: 10.1379/CSC-211.1

Figure Lengend Snippet: Effects of oxidative stress on protein kinase C (PKC)α-GFP and PKCβ-GFP in HeLa cells. (A, D) Effects of 100 μM H2O2; (B, E) histamine 100 μM in H2O2-pretreated cells; (C, F) effects of histamine 100 μM in untreated cells. HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum (FCS), in 75-cm2 flasks. The cells were seeded before transfection onto 24-mm glass coverslips and allowed to grow to 50% confluence. At this stage, transfection with 8 μg of the appropriate PKC plasmid DNA was carried out by Calcium phosphate technique as described previously (Chiesa et al 2001). Microscope analysis (and cell stimulation) were performed 36 h after transfection. To this end the medium was changed from DMEM + 10% FCS to KRB (125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM Na2HPO4, 5.5 mM glucose, 20 mM NaHCO3, 2 mM L-glutamine, 1 mM CaCl2, and 20 mM HEPES; pH 7.4). All the stimuli (H2O2, dithiothreitol [DTT], histamine, PMA, ascorbic acid) were added to the KRB at the time indicated in the figures. Images were recorded using a digital imaging system based on a Zeiss Axiovert 200 fluorescence microscope equipped with a back-illuminated charge-coupled device (CCD) camera (Roper Scientific, Trenton, NJ, USA). Rapid focussing in the z plane was guaranteed by excitation and emission filterwheels (Sutter Instrument Company, Novato, CA, USA) and piezoelectric motoring of the z stage (Physik Instrumente, GmbH & Co., Karlsruhe, Germany). The data were acquired and processed using the MetaMorph analyzing program (Universal Imaging Corporation, Downington, PA, USA). This allows the direct monitoring of fluorescence intensity. A high-resolution, 3D reconstruction of the distribution of a GFP chimera can be obtained with the technique of digital image restoration, also called deconvolution or deblurring. In brief, a series of 20 optical section images, spaced at 0.5-μm intervals through the depth of the cell, was acquired. Each optical section image was acquired in less than 1 second, and the entire through-focus series in less than 20 seconds. The image series then was deblurred using a constrained, iterative restoration algorithm that incorporates an empirically determined optical point spread function. The deblurred images then were used to visualize the 3D intracellular patterns. A more exhaustive description of this approach is presented in (Carrington et al 1995). In Figure 1Ci–v and Fi–ii, a larger magnification of the images is presented in the insets to allow a better appreciation of PKC translocation. The graphs (A′–F′) indicate the time course of plasma membrane translocation of PKC-GFP expressed as the increase in fluorescence ratio with respect to time zero (calculated as ratio of plasma membrane: average intracellular fluorescence). In all experiments, the images and traces are representative of at least 10 from 3 independent experiments, which gave similar results

Article Snippet: Rapid focussing in the z plane was guaranteed by excitation and emission filterwheels (Sutter Instrument Company, Novato, CA, USA) and piezoelectric motoring of the z stage (Physik Instrumente, GmbH & Co., Karlsruhe, Germany).

Techniques: Modification, Transfection, Plasmid Preparation, Microscopy, Cell Stimulation, Imaging, Fluorescence, Translocation Assay, Clinical Proteomics, Membrane