T H E J O U R N A L O F C E L L B I O L O G Y

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1 T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Feng et al., Figure S1. A modest elevation of disulfide-bonded K14 in primary mouse keratinocytes upon calcium switch. (A and B) The global disulfide bond staining in primary cultures of newborn mouse skin keratinocytes (A) and the localization of ER lumen (labeled using an antibody to recognize the marker protein disulfide isomerase [PDI]), where disulfide formation and isomerization of nascent proteins are catalyzed, thereby contributing to the perinuclear staining of disulfides labeled by maleimide (B). S-S, disulfide bonds; IgG, goat anti-rabbit IgG. Bar, 10 µm. (C) Disulfide-bonded K14 in primary mouse keratinocytes is modestly elevated upon calcium switch. Quantification of the total amount of disulfide-bonded K14 and K5 under low versus high calcium culture conditions achieved by densitometry-based analysis of disulfide-bonded versus monomeric keratins as reported in Fig. 1 A. Data were obtained from three independent experiments. Error bars represent standard deviations. Disulfide bonding and keratin filaments Feng and Coulombe S1

2 Figure S2. Intermolecular disulfide bonds involving K14WT, K14CF-C367, and K5WT. Keratin assemblies were subjected to high-speed sedimentation (150,000 g, 30 min), and pellet (Pel) and supernatant (Sup) fractions were loaded on nonreducing SDS-PAGE gels with or without being pretreated with a reducing agent followed by immunoblotting analysis. M, keratin monomer. Black lines indicate that intervening lanes have been spliced out. S2

3 Figure S3. Criteria used to assign keratin filament network organization to one of three categories: peripheral, pan-cytoplasmic, and perinuclear-concentrated networks. Using ImageJ, we determined the relative intensity of the fluorescence signal for keratin IFs (FI%) across the relative distance (RD) extending from the outer cell membrane (set as 0) to the nuclear envelope (set as 1), using transfected GFP-K14WT as an example. If keratin filaments (FI% > 50%) are distributed relatively evenly across the cytoplasm (RD = 0 1), such a network is designated pan-cytoplasmic (see A); if keratin filaments (FI% > 50%) are mainly concentrated in the perinuclear region (RD > 0.7), it is called perinuclear-concentrated (see B); finally, if keratin filaments (FI% > 50%) appear concentrated at the cell periphery (RD < 0.4), such a network is called peripheral (see C). For each graph, the 10 different color tracings represent 10 individual GFP-K14WT expressing keratinocytes. Disulfide bonding and keratin filaments Feng and Coulombe S3

4 Figure S4. Evidence that the existence of different phenotypes in Krt14 / cells expressing either K14WT or cysteine variants is not related to differences in transgene expression levels among individual cells. (A C) The K14 cysteine variants used in this study, e.g., K14CF, K14C367A, and K14CF-C367 (A), and fluorescence intensity comparison of GFP-K14WT versus GFP-K14CF (B), and GFP-K14CF-C367 expressing keratinocytes with (w/perin) or without (w/o pern) perinuclear-concentrated networks (C). Each symbol represents the mean fluorescence intensity of individual keratinocytes at seven time points (from 0 to 6 h) during movie recordings from a single experiment. n = 22 cells for GFP-K14WT, n = 22 cells for GFP-K14CF, n = 13 cells for GFP-K14CF- C367 w/perin, and n = 15 cells for GFP-K14CF-C367 w/o perin. S4

5 Figure S5. Enhanced nuclear and cellular movements manifested by GFP-K14CF-expressing keratinocytes. (A and B) Still images (A) and 2.5-dimentional views of z stacks of mobile GFP-K14CF expressing keratinocytes, derived from live movie recordings (B). Bar, 10 µm. (C F) Comparison of nuclear and whole cell movements obtained by tracking the position of the nucleus in GFP-K14WT (C), GFP-K14CF (D), and GFP-K14CF-C367 expressing (E) cells, as well as speed (F). More than 20 transfected cells were analyzed for each sample. Each color in C E and each symbol in F denote individual cells. Disulfide bonding and keratin filaments Feng and Coulombe S5

6 Video 1. GFP-K14WT-transfected K14 / mouse keratinocytes in primary culture. K14 / mouse keratinocytes were transfected with GFP-K14WT (green) and H2B-mCherry (red). Images were recorded using a laser scanning confocal microscope (LSM780-FCS, Carl Zeiss). Recording intervals were 5 min and the whole recording time was 6 h. Video 2. GFP-K14CF-transfected K14 / mouse keratinocytes in primary culture showing a stationary behavior. K14 / mouse keratinocytes were transfected with GFP-K14CF (green) and H2B-mCherry (red). Images were recorded using a laser scanning confocal microscope (LSM780-FCS; Carl Zeiss). Recording intervals were 5 min and the whole recording time was 6 h. Video 3. GFP-K14CF transfected K14 / mouse keratinocytes in primary culture showing a motile behavior. K14 / mouse keratinocytes were transfected with GFP-K14CF (green) and H2B-mCherry (red). Images were recorded using a laser scanning confocal microscope (LSM780-FCS; Carl Zeiss). Recording intervals were 5 min and the whole recording time was 6 h. Video 4. GFP-K14CF-C367 transfected K14 / mouse keratinocytes in primary culture showing perinuclear-concentrated networks. K14 / mouse keratinocytes were transfected with GFP-K14CF-C367 (green) and H2B-mCherry (red). Images were recorded using a laser scanning confocal microscope (LSM780-FCS; Carl Zeiss). Recording intervals were 5 min and the whole recording time was 6 h. Video 5. GFP-K14CF-C367 transfected K14 / mouse keratinocytes in primary culture showing a lack of stable perinuclearconcentrated networks. K14 / mouse keratinocytes were transfected with GFP-K14CF-C367 (green) and H2B-mCherry (red). Images were recorded using a laser scanning confocal microscope (LSM780-FCS; Carl Zeiss). Recording intervals were 5 min and the whole recording time was 6 h. S6