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PMC1193645

14699080

20020509f1

Figure 1. Spontaneous EAE development in wild-type and STAT1-deficient mice. (A) TCR + STAT1 −/− H-2 b/u mice and their TCR + STAT1 +/− H-2 b/u littermates (30 animals/group) were monitored for 6 mo and the spontaneous development of EAE (level 5) was recorded. (B) Same as in A, but TCR + STAT1 −/− H-2 u/u mice and their TCR + STAT1 +/− H-2 u/u littermates (20 animals/group) were used.

CC BY-NC-SA

no

2022-01-13 08:52:49

J Exp Med. 2004 Jan 5; 199(1):25-34

PMC1193645

14699080

20020509f2

Figure 2. Flow cytometric analysis of wild-type, asymptomatic, or paralyzed STAT1-deficient mice. (A) Cells derived from lymph nodes of wild-type, asymptomatic, or paralyzed STAT1-deficient animals were stained for CD4, CD69, and Vβ8. Horizontal axis represents Vβ8 staining, vertical axis corresponds to CD4 (top) or CD69 (bottom) staining of CD8 − cells. Cells displayed in lower panels are CD4 + . (B) Splenocytes derived from wild-type, asymptomatic, or paralyzed STAT1-deficient animals were stimulated with 50 ng/ml PMA/500 ng/ml ionomycin for 4 h (10 μg/ml brefeldin A was added during the last 2 h) and stained for CD4, CD8, and intracellular IFN-γ. CD4 staining (horizontal axis) and IFN-γ production (vertical axis) of CD8 − cells is shown.

CC BY-NC-SA

no

2022-01-13 08:52:49

J Exp Med. 2004 Jan 5; 199(1):25-34

PMC1193645

14699080

20020509f3

Figure 3. Immunohistological analysis of CNS tissue from asymptomatic and paralyzed STAT1-deficient mice. Brains and spines were removed from asymptomatic or paralyzed (level 5) STAT1-deficient mice and fixed in formaldehyde. Paraffin-embedded sections were stained with hematoxylin and eosin.

CC BY-NC-SA

no

2022-01-13 08:52:49

J Exp Med. 2004 Jan 5; 199(1):25-34

PMC1193645

14699080

20020509f4

Figure 4. Proliferation of wild-type and STAT1-deficient CD4 + cells. (A) CD4 + cells were purified from the spleens of wild-type or STAT1-deficient mice as described in Materials and Methods, and proliferation was assessed by thymidine incorporation in response to stimulation with 1.0 μg/ml Con A in the presence of mitomycin C–treated APCs. (B) CD4 + cells were purified from the spleens of TCR + STAT1 −/− H-2 b/u mice and their TCR + STAT1 +/− H-2 b/u littermates, and proliferation was measured by thymidine incorporation. Mitomycin C–treated APCs were generated from STAT1 +/− H-2 b/u animals and preincubated with 100 μg/ml MBP as indicated.

CC BY-NC-SA

no

2022-01-13 08:52:49

J Exp Med. 2004 Jan 5; 199(1):25-34

PMC1193645

14699080

20020509f5

Figure 5. (A and B) Flow cytometric analysis of thymocytes in wild-type and STAT1-deficient mice. Cells derived from thymi of wild-type or STAT1-deficient animals were stained for CD4, CD8, and CD25. The ratio of CD4 + CD8 − CD25 + cells as the percentage of total CD4 + CD8 − cells is shown.

CC BY-NC-SA

no

2022-01-13 08:52:49

J Exp Med. 2004 Jan 5; 199(1):25-34

PMC1193645

14699080

20020509f6ab

Figure 6. Effect of wild-type CD4 + CD25 + cells on the proliferation of STAT1 −/− CD4 + cells. (A) CD4 + cells were purified from the spleens of wild-type or STAT1-deficient mice as described in Materials and Methods. Wild-type or STAT1-deficient CD4 + cells were either incubated separately, mixed in a 1:1 ratio, or wild-type cells were CD25 depleted before mixing 1:1 with STAT1-deficient cells as indicated. Proliferation was then assessed by thymidine incorporation in response to stimulation with 1.0 μg/ml Con A in the presence of mitomycin C–treated APCs. (B) Same as in A, but cells were stimulated with 50 ng/ml anti-CD3 antibody in combination with 50 U/ml human rIL-2. (C) CD4 + cells were purified from the spleens of wild-type or STAT1-deficient mice as described in Materials and Methods. STAT1-deficient cells were labeled with CFSE and their proliferation in response to 1.0 μg/ml Con A in the presence of either unlabeled wild-type CD4 + cells or unlabeled CD25-depleted wild-type CD4 + cells (1:1 ratio) was determined by flow cytometry. (D) CD4 + cells were purified from the spleens of wild-type mice, depleted of CD25 + cells, and labeled with CFSE. Proliferation in response to 1.0 μg/ml Con A in the presence of either unlabeled STAT1 −/− CD4 + cells or unlabeled CD25-depleted STAT1 −/− CD4 + cells (1:1 ratio) was determined by flow cytometry.

CC BY-NC-SA

no

2022-01-13 08:52:49

J Exp Med. 2004 Jan 5; 199(1):25-34

PMC1193645

14699080

20020509f7

Figure 7. STAT1-deficient CD4 + CD25 + cells fail to suppress T cell proliferation. CD4 + CD25 + and CD4 + CD25 − cells were purified from spleens as described in Materials and Methods. Wild-type or STAT1-deficient CD4 + CD25 − cells were labeled with CFSE and their proliferation in response to 1.0 μg/ml Con A in the absence or presence of either unlabeled wild-type or STAT1-deficient CD4 + CD25 + cells was determined by flow cytometry (ratios of CD4 + CD25 − /CD4 + CD25 + cells were 1:0.1, 1:0.2, 1:0.4, and 1:0.8 in lanes 3–6 and 10–13, respectively). A representative of three independent experiments is shown.

CC BY-NC-SA

no

2022-01-13 08:52:49

J Exp Med. 2004 Jan 5; 199(1):25-34

PMC1237012

15583013

20040915f1

Figure 1. Restriction maps of CD73 genomic DNA, targeting vector, and recombined allele. (A) CD73 genomic DNA, including exon 3 and intronic sequences used to construct the short (1 kb) and long arms (4.2 kb) of the targeting vector. (B) CD73 targeting vector showing the antisense orientation of the CD73 sequences relative to the neo cassette. (C) Recombined, gene-targeted CD73 allele. The flanking probe used for Southern blots is shown, as are the positions of the PCR primers (arrows) used for screening the ES cell clones. (D) Southern blot of genomic DNA from Cd73 +/+ , Cd73 −/+ , and Cd73 −/− mice. Genomic DNA was purified from mouse tails, digested with BamHI, and subjected to Southern blotting by conventional methods. The probe was from intronic sequence 3′ to the long arm as shown in C. The wild-type BamHI fragment is 8.9 kb, whereas that from the gene-targeted mice is 7.4 kb. (E) Northern blot of kidney RNA from Cd73 +/+ and Cd73 −/− mice. Triplicate Northern blots were performed with 15 μg of kidney RNA in each lane. Blots were hybridized to the following probes: exon 2 (5′ probe), exon 3, or exon 4 (3′ probe). Ethidium bromide staining was used to confirm equal RNA loading.

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2022-01-13 08:53:02

J Exp Med. 2004 Dec 6; 200(11):1395-1405

PMC1237012

15583013

20040915f2

Figure 2. CD73 expression and function on leukocytes from Cd73 +/+ and Cd73 −/− mice. CD73 expression was evaluated on leukocytes from lymph node, spleen, peripheral blood, bone marrow, and thymus from Cd73 +/+ (left) and Cd73 −/− (right) mice with monoclonal antibody TY/23 + PE–goat anti–rat IgG. Staining with an isotype-matched control antibody is shown in the shaded histograms.

CC BY-NC-SA

no

2022-01-13 08:53:02

J Exp Med. 2004 Dec 6; 200(11):1395-1405

PMC1237012

15583013

20040915f3

Figure 3. Vascular leakage in Cd73 -deficient mice in vivo. Cd73 −/− mice (black bars) and age-, weight-, and gender-matched littermate controls (white bars) were administered intravenous Evan's blue (0.2 ml of 0.5% in PBS per mouse) and exposed to room temperature air (A) or normobaric hypoxia (B, 8% O 2 , 92% N 2 ) for 4 h. Animals were killed, and the colon (Co), lung (Lg), liver (Lv), muscle (Mu), heart (Ht), kidney (Kd), and brain (Br) were harvested. Evan's blue concentrations in organs were quantified as described in Materials and Methods. Data are expressed as mean ± SD Evan's blue OD/50 mg wet tissue and are pooled from four to six animals per condition, where * indicates P < 0.025 between Cd73 +/+ and Cd73 −/− mice and # indicates P < 0.025 between hypoxia and normoxia. (C) Images of abdominal dissections from wild-type and Cd73 −/− mice subjected to normoxia and hypoxia for 4 h.

CC BY-NC-SA

no

2022-01-13 08:53:02

J Exp Med. 2004 Dec 6; 200(11):1395-1405

PMC1237012

15583013

20040915f4

Figure 4. Influence of CD73 inhibition on vascular leakage in vivo. Age-, weight-, and gender-matched mice were administered APCP (20 mg/kg i.p.) or an equal volume of PBS followed by intravenous Evan's blue solution (0.2 ml of 0.5% in PBS per mouse) and exposed to room temperature air (A) or to normobaric hypoxia (B, 8% O 2 , 92% N 2 ) for 4 h. Animals were killed and the colon (Co), lung (Lg), liver (Lv), muscle (Mu), heart (Ht), kidney (Kd), and brain (Br) were harvested. Evan's blue concentrations in organs were quantified as described in Materials and Methods. Data are expressed as mean ± SD Evan's blue OD/50 mg wet tissue and are pooled from four to six animals per condition where * indicates P < 0.05 in comparisons between APCP and PBS, and # indicates P < 0.025 between hypoxia and normoxia. (C) HPLC analysis of CD73 5′-NT enzyme activity (conversion of 1 mM E-AMP to E-Ado) in serum harvested at 4 h from normoxic animals administered −APCP (top) or +APCP (bottom).

CC BY-NC-SA

no

2022-01-13 08:53:02

J Exp Med. 2004 Dec 6; 200(11):1395-1405

PMC1237012

15583013

20040915f5

Figure 5. Vascular leakage and reconstitution of Cd73 −/− mice with 5′-NT. Cd73 −/− mice (B and D) and age-, weight-, and gender-matched littermate controls (A and C) were administered 5′-NT purified from C. atrox venom (500 U/kg i.p.; black bars) or PBS (white bars) followed by intravenous Evan's blue solution (0.2 ml of 0.5% in PBS per mouse) and exposed to room temperature air (A and B) or normobaric hypoxia (C and D, 8% O 2 , 92% N 2 ) for 4 h. Animals were killed and the colon (Co), lung (Lg), liver (Lv), muscle (Mu), heart (Ht), kidney (Kd), and brain (Br) were harvested. Evan's blue concentrations in organs were quantified as described in Materials and Methods. Data are expressed as mean ± SD Evan's blue OD/50 mg wet tissue and are pooled from four to six animals per condition where, in comparisons between 5′-NT and PBS, * indicates P < 0.05 and # indicates P < 0.025.

CC BY-NC-SA

no

2022-01-13 08:53:02

J Exp Med. 2004 Dec 6; 200(11):1395-1405

PMC1237012

15583013

20040915f6

Figure 6. Influence of adenosine receptor antagonists on hypoxia-induced vascular leakage. Wild-type mice were administered either PBS (Control), the A 2B receptor antagonist MRS1754 (1 mg/kg i.p. plus 1 mg/kg s.c.) or the A 2A receptor antagonist ZM241385 (1 mg/kg i.p. plus 1 mg/kg s.c.) followed by intravenous Evan's blue solution (0.2 ml of 0.5% in PBS per mouse) and exposed to room air (black bars) or normobaric hypoxia (gray bars, 8% O 2 , 92% N 2 ) for 4 h. Animals were killed, and lungs were harvested. (A) Evan's blue concentrations in organs were quantified as described in Materials and Methods. Data are expressed as mean ± SD Evan's blue OD/50 mg wet tissue and are pooled from four animals per condition where * indicates P < 0.01 between normoxia and hypoxia and # indicates P < 0.01 between treatment and control. (B) Assessment of lung water content. Data are expressed as mean ± SD mg H 2 O/mg dry tissue, and are pooled from four animals per condition where, in comparisons between hypoxia and normoxia, * indicates P < 0.01, # indicates P < 0.05 between treatment and control, and ## indicates P < 0.025 between treatment and control.

CC BY-NC-SA

no

2022-01-13 08:53:02

J Exp Med. 2004 Dec 6; 200(11):1395-1405

PMC1237012

15583013

20040915f7

Figure 7. Influence of the adenosine receptor agonist NECA on vascular leakage of Cd73 −/− mice. Cd73 −/− mice (B and D) and age-, weight-, and gender-matched littermate controls (A and C) were administered the adenosine analogue NECA (0.1 mg/kg i.p. plus 0.1 mg/kg s.c., black bars) or PBS (white bars) followed by intravenous Evan's blue solution (0.2 ml of 0.5% in PBS per mouse) and exposed to room temperature air (A and B) or normobaric hypoxia (C and D, 8% O 2 , 92% N 2 ) for 4 h. Animals were killed, and the colon (Co), lung (Lg), liver (Lv), muscle (Mu), heart (Ht), kidney (Kd), and brain (Br) were harvested. Evan's blue concentrations in organs were quantified as described in Materials and Methods. Data are expressed as mean ± SD Evan's blue OD/50 mg wet tissue and are pooled from four to six animals per condition where * indicates P < 0.025 and # indicates P < 0.01 between NECA and PBS.

CC BY-NC-SA

no

2022-01-13 08:53:02

J Exp Med. 2004 Dec 6; 200(11):1395-1405

PMC1237012

15583013

20040915f8

Figure 8. Characterization of lungs from Cd73 −/− mice. Wild-type (A and D) or Cd73 −/− (B and C) mice were subjected to normoxia (A and B) or hypoxia (C and D). Whole lungs were fixed with 10% formalin at total lung capacity, sectioned, and stained with hematoxylin and eosin. (A) A representative image of a wild-type control animal (magnification, 100). (C) Perivascular interstitial edema (line arrows) and epithelial disruption (block arrow) in Cd73 −/− hypoxic mice (magnification, 100). (D) Subtle perivascular interstitial edema (line arrow) associated with wild-type hypoxic mice (magnification, 100). (E) Assessment of lung water content in normoxia (black bars) and hypoxia (gray bars) in the presence and absence of NECA administration (0.1 mg/mg i.p. plus 0.1 mg/kg s.c.). Data are expressed as mean ± SD mg H 2 O/mg dry tissue and are pooled from three to four animals per condition where, in comparisons between hypoxia and normoxia, * indicates P < 0.025 and, in comparisons between NECA and PBS, # indicates P < 0.025.

CC BY-NC-SA

no

2022-01-13 08:53:02

J Exp Med. 2004 Dec 6; 200(11):1395-1405

PMC1237122

15699070

20041577f1

Figure 1. LN1 nonproductive allele structure. Configurations of wild type and LN1 nonproductive (ΔD) IgH loci are depicted together with proposed intermediates. Nucleotides inserted during the ΔD rearrangement are highlighted in italics and nucleotides that are lost in the process of rearrangement are shaded. The ΔD rearrangement may be the result of incorrect resolution of the RAG–DNA complex that results in J H 1 joining the sequence upstream of the DFL16.1 instead of the DFL16.1 in an inverted orientation.

CC BY-NC-SA

no

2022-01-13 09:00:15

J Exp Med. 2005 Feb 7; 201(3):341-348

PMC1237122

15699070

20041577f2

Figure 2. ΔD/ΔD mice generate all peripheral B cell subsets with a low rate of B cell production. Representative flow cytometric analyses of lymphocytes in spleen (A), bone marrow (B), and peritoneal cavity (C) of ΔD/ΔD and WT mice. Shown are cells in the “lymphocyte gate” unless additional gates are specified. Numbers within the FACS plots indicate the percent of cells that fall into a given gate. For spleen (A) and bone marrow (B), average values and standard deviations are shown for different B cell subsets. ΔD/ΔD mice are represented by closed bars, and WT mice by open bars. n = 6 for 5-wk-old ΔD/ΔD mice, n = 4 for 5-wk-old WT mice, n = 3 for 6-mo-old ΔD/ΔD mice, and n = 2 for 6-mo-old controls. Representative FACS plots shown in the figure are for 6-mo-old mice.

CC BY-NC-SA

no

2022-01-13 09:00:15

J Exp Med. 2005 Feb 7; 201(3):341-348

PMC1237122

15699070

20041577f3

Figure 3. The ΔD allele cannot compete with the WT allele in either B cell generation or antibody production. FACS analysis of CD19-gated splenocytes from 5-mo-old ΔD/ΔD, (BALB/c × ΔD)F1, (BALB/c × C57BL/6)F1, C57BL/6, and BALB/c mice for the expression of IgM of a and b allotypes. The ΔD allele is of the b allotype, the IgH loci of BALB/c and C57BL/6 mice are of the a and b allotype, respectively. Bar graphs below the FACS plots summarize the ELISA data with serum IgM levels plotted in μg/ml for 10-wk-old mice ( n = 2 for each group). Gray bars represent IgM of the a allotype and black bars IgM of the b allotype.

CC BY-NC-SA

no

2022-01-13 09:00:15

J Exp Med. 2005 Feb 7; 201(3):341-348

PMC1237122

15699070

20041577f4

Figure 4. Analysis of IgH V region joints derived from B cells in ΔD mice. (A) Alignment of joints amplified from the RNA of 2-wk-old ΔD/ΔD and ΔD/JHT mice. The joints are shown from the codon immediately 5′ of the second cystein (position 104) of the V H gene and extending to the conserved glycine of the J H region. Sequences labeled with an asterisk use the putative D H element, DST4.2 (underlined). The sequences were analyzed using the http://www.DNAPLOT.de , http://www.imgt.cines.fr , or http://www.ncbi.nlm.nih.gov/igblast websites. In the analysis of bulk-sorted cells, some sequences were found repeatedly, as indicated by the superscripts next to the sequence numbers. As we did not observe repeated sequences in the single cell analyses, we consider the repeats in the bulk analysis an artifact resulting from the high number of amplification cycles. Two sets of sequences (1, 11 and 30, 31, 32) may represent hybrid sequences generated in the course of gene amplification by PCR (reference 27 ). Sequences were submitted through http://www.ncbi.nih.gov/Genbank/index.html in a consistent order (GenBank/EMBL/DDBJ accession nos. AY841948 , AY841949 , AY841950 , AY841951 , AY841952 , AY841953 , AY841954 , AY841955 , AY841956 , AY841957 , AY841958 , AY841959 , AY841960 , AY841961 , AY841962 , AY841963 , AY841964 , AY841965 , AY841966 , AY841967 , AY841968 , AY841969 , AY841970 , AY841971 , AY841972 , AY841973 , AY841974 , AY841975 , AY841976 , AY841977 , AY841978 , AY841979 ). (B) Analysis of joints from single cell sorted and bulk sorted or MACS B cells from 5- and 10-wk-old ΔD/ΔD or WT mice. Because of space limitations, only their productive versus nonproductive status is listed. (C) CDR3 length comparison of V H J H joints (excluding sequences using DST4.2) from ΔD/ΔD and ΔD/JHT B cells and V H D H J H joints from WT B cells isolated from 2-, 5-, and 10-wk-old mice. Gray bars represent average number of nucleotides in the CDR3 defined as starting after the cysteine in the 3′ end of the V H and ending with the last nucleotide before the conserved tryptophan of J H . Error bars represent standard deviations. To demonstrate that the difference in the CDR3 length of the joints from ΔD/ΔD and WT B cells is due to the absence of D H elements and only a single round of N and P nucleotide addition, the average length of D H sequence in WT V H D H J H joints (white bars) plus that of N/P nucleotides added in a single round (black bars) are shown on top of the CDR3 values for ΔD/ΔD sequences. The first group of bars represents a mix of sequences amplified from cDNA of 2-wk-old mice with a natural distribution of J H usage. The second and third group is from DNA of 5-wk-old mice sequenced using a J H 4 or J H 2 primer, respectively. Because J H element length varies, different J H elements contribute differently to overall CDR3 length. The last bar gives the average and standard deviation for sequences derived from DNA of single cells of a 10-wk-old mouse using J H 2 primer. Sequences from appropriately age-matched WT mice were are not available. The average D H element length in V H D H J H joints was calculated from the number of nucleotides of D H origin in the WT sequences of the corresponding group. To approximate the average number of nucleotides per one round of N/P nucleotide addition, the N/P nucleotides at the D H J H and V H D H border in the WT joints of a corresponding group were added and divided by the number of sequences and by a factor of two.

CC BY-NC-SA

no

2022-01-13 09:00:15

J Exp Med. 2005 Feb 7; 201(3):341-348

PMC1237122

15699070

20041577f5

Figure 5. Absence of newly generated B cells expressing the ΔD allele in heterozygous mutant mice. Immature and mature B cells in the bone marrow of 10-mo-old (BALB/c × ΔD)F1 mice were compared with those of 5-mo-old (BALB/c × C57BL/6)F1, BALB/c, and C57BL/6 mice ( n = 2 for each group) for expression of either IgM a or IgM b . The ΔD allele is of the b allotype, and the IgH loci of BALB/c and C57BL/6 mice are of the a and b allotype, respectively. The gated B220 lo IgD − population contains B cell progenitors and immature, surface IgM + B cells, which are analyzed for IgM allotype expression (middle). The gated B220 hi IgD + population represents mature B cells, which are analyzed for IgM allotype expression (bottom).

CC BY-NC-SA

no

2022-01-13 09:00:15

J Exp Med. 2005 Feb 7; 201(3):341-348

PMC1255917

11470818

0104043f1

Figure 1. Relative replication timing of DHFR and β -globin loci. (A) Asynchronously growing CHO cells were pulse labeled with BrdU and hybridized in situ with digoxigenin-labeled DHFR cosmid cSc26 and biotin-labeled β-globin phage λJHC2. Sites of hybridization were visualized with FITC-conjugated antidigoxigenin antibodies (DHFR, green) and Texas red avidin (β-globin, red). BrdU label was detected with AMCA-labeled anti-BrdU antibodies (blue). In the image shown, BrdU foci are not visible because nuclei were additionally stained with DAPI (blue) to highlight the entire nucleus. (B–D) The number of DHFR and β-globin singlets and doublets per nucleus was evaluated. (B) The percentage of total alleles scored that displayed doublets for each locus was calculated. (C) The percentage of nuclei that displayed more doublets for one locus than the other was calculated. (D) The percentage of nuclei that showed doublets for both alleles of one locus and singlets for both alleles of the other locus was calculated. (C and D) Schematic representations of the arrangement of the DHFR (dot) and β-globin (x) alleles are included. The mean values ± SEM are shown for three independent experiments in which >100 BrdU-positive nuclei each were scored. All sets of data in this report include hybridizations in which both DHFR and β-globin probes were labeled with both biotin and digoxigenin and used in either combination, to control for any differences in labeling and detection of different nucleotide analogues. Only nuclei showing clear signals for both DHFR and β-globin loci were scored.

CC BY-NC-SA

no

2022-01-13 07:19:29

J Cell Biol. 2001 Jul 23; 154(2):283-292

PMC1255917

11470818

0104043f2

Figure 2. The CHO β -globin locus is replicated during the stage of peripheral DNA synthesis. (A) Examples of early (type I/II), middle (type III), and late (type and type V) replication patterns, revealed by pulse labeling CHO AA8 cells with BrdU and staining with fluorescent anti-BrdU antibodies. (Top) Shows cells fixed with ethanol and stained only for anti-BrdU, as described ( Dimitrova and Gilbert, 1999b ). (Bottom) Shows the pattern of anti-BrdU staining with cells that were first subjected to FISH. Conditions used for FISH distort the pattern of BrdU staining; however, it is still possible to distinguish the different pattern types. (B) CHO AA8 cells were synchronized in mitosis and released into medium containing aphidicolin to accumulate cells at the G1/S border. Cells were then released from the G1/S block, pulse labeled with BrdU at various times thereafter, and stained with anti-BrdU antibodies as in A (top). Shown are the percentage of BrdU-positive cells that exhibited early (▪), middle (▴), or late (•) replication patterns at each time point. (C) Aliquots of the same cells from B were hybridized with β-globin and DHFR probes by FISH, and the percentage of doublet alleles for each locus was determined. Results in B and C show the means ± SEM (when >2) for three independent experiments in which >100 nuclei each were scored. (D and E) Cells synchronized as in B were subjected to FISH with either a DHFR (D) or β-globin (E) probe and subsequently stained with anti-BrdU antibodies as in A (bottom). The percentage of doublet alleles was scored as a function of the early (▪), middle (▴), or late (•) BrdU patterns. The dashed lines for middle- and late-replication patterns indicate the time of appearance of these patterns during S phase (i.e., middle patterns first appeared between 2.5 and 4.5 h, and late patterns first appeared between 4.5 and 6.5 h).

CC BY-NC-SA

no

2022-01-13 07:19:29

J Cell Biol. 2001 Jul 23; 154(2):283-292

PMC1255917

11470818

0104043f3

Figure 3. Colocalization of β -globin versus DHFR doublets with sites of DNA synthesis during middle to late S phase. CHO AA8 cells pulse labeled with BrdU were subjected to FISH with either a β-globin or a DHFR probe (red) and then stained with anti-BrdU antibodies (green). Analysis was concentrated on nuclei that displayed type III or IV BrdU patterns (described in legend to Fig. 2 ). (A) (Top) Show examples of merged confocal images. (Bottom) Show the same images after computer-assisted colocalization analysis. After background subtraction, those pixels still showing colocalization of red and green signals were colored in yellow, whereas pixels showing no colocalization were colored in white. (B) Sections of the nuclear periphery from exemplary nuclei showing the proximity of β-globin doublets to the peripheral heterochromatin. (C) The percentage of doublets within type III and IV nuclei that colocalized with BrdU signal was scored. Data were scored independently for doublets found either at the periphery (defined as <1 μm from the edge of DAPI staining) or the interior of the nucleus.

CC BY-NC-SA

no

2022-01-13 07:19:29

J Cell Biol. 2001 Jul 23; 154(2):283-292

PMC1255917

11470818

0104043f4

Figure 4. Late replication of the β -globin gene locus in CHO cells is established 1–2 h after mitosis. (A) CHO AA8 cells were synchronized in mitosis and released into G1 phase. At the indicated times thereafter, cells were pulse labeled with BrdU, stained with anti-BrdU antibodies, and the percentage of BrdU positive nuclei was scored. (B) Nuclei isolated either at 1, 2, or 3 h after mitosis were introduced into Xenopus egg extracts. Aliquots of these nuclei were then subjected to FISH analysis at 30, 60, and 120 min thereafter. The percentage of total DHFR or β-globin alleles displaying doublets was calculated for >100 nuclei per time point. Shown are the means ± SEM for three independent experiments. (C–E) Results shown in B for 30, 60, and 120 min in vitro were averaged together and displayed by the same three methods described in the legend to Fig. 1, B–D .

CC BY-NC-SA

no

2022-01-13 07:19:29

J Cell Biol. 2001 Jul 23; 154(2):283-292

PMC1255917

11470818

0104043f5

Figure 5. The repositioning of type III sequences and the peripheral localization of the β -globin locus are completed 1–2 h after mitosis. (A) Asynchronous cultures of CHO AA8 cells were pulse labeled with BrdU for 30 min. Metaphase cells were harvested 3.5 h thereafter, creating populations of cells in which late-replicating sequences were tagged with BrdU (∼1/3 of which were type III). At 1, 2, and 3 h after release into G1 phase, cells were fixed and stained with anti-BrdU antibody, and the percentage of nuclei displaying a type III spatial pattern, represented by peripheral and nucleolar BrdU staining, was calculated. (B) Nuclei were isolated from CHO AA8 cells synchronized at 1, 2, or 3 h after mitosis and introduced into Xenopus egg extracts. At the indicated time points, reactions were pulse labeled in vitro with biotin-11-dUTP, and nuclei were stained with Texas red streptavidin. The percentage of nuclei from each time point that displayed either early (many internal punctate foci) or middle (peripheral and nucleolar DNA synthesis) replication patterns was calculated. (C and D) Asynchronously growing cultures (Asyn.), as well as cells synchronized at 1, 2, or 3 h after metaphase, were subjected to FISH with DHFR or β-globin probes and then stained with an anti-lamin A/C antibody. Confocal images were collected and the distance from each allele to the nuclear lamina was measured and divided by the radius of the nucleus at its widest point. (C) Examples of images found for either DHFR or β-globin at either 1 or 2 h after mitosis. A copy of each image is shown to its immediate right, with white lines to denote the measurements taken for the diameter and the shortest distance from the FISH signal to the nuclear envelope. The lamina was used as an indicator of the nuclear periphery rather than DAPI (as in Fig. 1 ) due to the occasional invaginations of the nuclear envelope that are not always detectable with the use of DNA dyes. An example of such an invagination is shown (2, a and b). Two β-globin alleles at the periphery, appearing as yellow FISH foci, which would be scored as a distance of zero, are also shown (3a and 3b). (D) Box plot representing the shortest distance between DHFR or β-globin alleles and the nuclear lamina. Data for each cell population were collected from ≥37 nuclei. Horizontal bars represent the 10th, 25th, 50th (median), 75th, and 90th percentiles, and the p- values for each pair of samples are shown. This type of plot indicates that 50% of the population is found between the 25th and 75th percentiles, and is represented as a box. Gray boxes denote DHFR alleles, and white boxes denote β-globin alleles.

CC BY-NC-SA

no

2022-01-13 07:19:29

J Cell Biol. 2001 Jul 23; 154(2):283-292

PMC1255922

11535615

0104099f1

Figure 1. Chk1 and Chk2 activation in response to HU and IR. (A) HeLa cell lysate (50 μg) subjected to SDS-PAGE and immunoblotted with anti-Chk1 antibodies. (B) Kinase assays in the presence of indicated concentrations of UCN-01 performed on Chk1 IPs from HeLa cell lysates. (C) HeLa cells ± caffeine (5 mM), were treated with hydroxyurea (+HU, 2 mM) or irradiated (10 Gy, +IR) as indicated. Cells were harvested, lysed and Chk1 IP kinase assays performed. (D) Lysates from cells treated as in C were subjected to SDS-PAGE and immunoblotted for Chk1. (E) Kinase assays performed on Chk2 IPs from lysates obtained as in C. (F) Immunoblot analysis of Chk2 following treatments indicated in E.

CC BY-NC-SA

no

2022-01-13 07:19:32

J Cell Biol. 2001 Sep 3; 154(5):913-924

PMC1255922

11535615

0104099f2

Figure 2. S phase activation of Chk1 and Chk2 is independent of ATM. (A–D) ATM-null (AT221JE-T) cells were untreated, treated with HU (+HU, 2 mM) or irradiated (10 Gy, +IR) as indicated. (A) Cells were lysed, and kinase assays performed on Chk1 IPs. (B) Lysates subjected to SDS-PAGE and immunoblotted for Chk1. (C) Kinase assays performed on Chk2 IPs. (D) Lysates subjected to SDS-PAGE and immunoblotted for Chk2. (E-H) AT221JE-T cells containing either vector alone (−ATM) or vector expressing ATM (+ATM) were irradiated (10 Gy, +IR) or not as indicated, and lysates prepared. (E) Kinase assays performed on immunoprecipitated Chk1. (F) Lysates subjected to SDS-PAGE and immunoblotted for Chk1. (G) Kinase assays were performed on immunoprecipitated Chk2. (H) Lysates subjected to SDS-PAGE and immunoblotted for Chk2.

CC BY-NC-SA

no

2022-01-13 07:19:32

J Cell Biol. 2001 Sep 3; 154(5):913-924

PMC1255922

11535615

0104099f3ad

Figure 3. Activation of Chk1 and Chk2 requires DNA replication arrest. Metaphase HeLa cells collected by nocodazole treatment and mitotic shake-off were released into fresh medium +/−HU. 0.5 h before harvest, at indicated times after nocodazole release, cells were pulse labeled with BrdU, and aliquots removed for flow cytometry (A–D), lysis and IP kinase assays (E and H) and immunoblotting (F and G). (A–D) FACS ® profiles and cell cycle position are shown for cells maintained in the absence (-HU, A and B) and presence (+HU, C and D) of HU (2 mM). (E) Chk1 IP kinase assays performed on lysates from asynchronous cycling cells (AS, grey bars), cells progressing through S phase (white bars) and arrested in early S phase (black bars). (F) Cell lysates as in E above subjected to SDS-PAGE and immunoblotted for Chk1. (G) Cell lysates as in E above subjected to SDS-PAGE and immunoblotted for Chk2. (H) Chk2 IP kinase assays performed on lysates from cells as in E.

CC BY-NC-SA

no

2022-01-13 07:19:32

J Cell Biol. 2001 Sep 3; 154(5):913-924

PMC1255922

11535615

0104099f4ab

Figure 4. Differential inactivation of Chk1 and Chk2 following release from replication block. (A) Asynchronously growing HeLa cells were treated or not for 24 h with 2 mM HU or 5 μg/ml aphidicolin and lysates prepared. Kinase assays were performed on immunoprecipitated Chk1 and Chk2 (upper panels) as indicated. Lysates were subjected to SDS-PAGE and immunoblotted for Chk1 and Chk2 (lower panels). (B) Asynchronously growing AT221JE-T cells expressing ATM were treated with 2 mM HU for 24 h, released, and at times indicated pulse labeled with BrdU. Cells were fixed, stained with DAPI and anti-BrdU antibodies, and examined by indirect immunofluorescence microscopy. Identical results were observed with AT cells transfected with empty vector (not shown). (C and D) AT221JE-T cells containing either vector alone ( − ATM) or vector expressing ATM (+ATM) were treated for 24 h with HU, and then released into fresh medium. (C) At the times indicated, lysates were prepared, subjected to SDS-PAGE, and immunoblotted for Chk1 and Chk2. (D) At times indicated, Chk1 (upper panels) and Chk2 (lower panels) IP kinase assays were performed on lysates from AT221JE-T cells containing vector alone (black bars) or vector expressing ATM (white bars) treated as above.

CC BY-NC-SA

no

2022-01-13 07:19:32

J Cell Biol. 2001 Sep 3; 154(5):913-924

PMC1255922

11535615

0104099f5

Figure 5. UCN-01 blocks Chk1 autophosphorylation in vitro and Chk1 hyperphosphorylation in vivo, with no effect on ATR kinase in vitro or S phase activation of Chk2 in vivo. (A) Chk1 autophosphorylation was measured by incorporation of 32 P from labeled ATP in the presence of 0 (lane 1), 10 nM (lane 2) 50 nM (lane 3), and 300 nM UCN-01 (lane 4) followed by SDS-PAGE and autoradiography. (B) HeLa and CHO cells were treated +/− HU (2 mM, 15 h) in the presence or absence of 300 nM UCN-01 and lysates analyzed for Chk1 by SDS-PAGE and immunoblotting. Immunoblot is overexposed to show all reduced mobility forms of Chk1 on HU treatment. (C) SDS-PAGE and autoradiography of kinase-dead Chk1 following its phosphorylation by mock (lane 1), purified ATR in the presence of 0 (lane 2), 0.5 ng/μl (lane 3), 2 ng/μl (lane 4), and 4 ng/μl DNA (lane 5) and (D) DNA-dependent phosphorylation of kinase-dead Chk1 by purified ATR in the presence of 0 (lane 1), 10 nM (lane 2) 50 nM (lane 3), and 300 nM UCN-01 (lane 4). (E) HeLa cells treated +/−HU as above in the presence or absence of 300 nM UCN-01 or 5 mM caffeine, and lysates analyzed for Chk1 by SDS-PAGE and immunoblotting. Samples in lanes 1–3 are identical to upper panel of B but at lower exposure to resolve distinct forms of Chk1 after indicated treatments. (F) HeLa cells treated +/− HU as above in presence or absence of 300 nM UCN-01 as indicated, were lysed and Chk2 analysed by IP kinase assay (upper panel) or by SDS-PAGE and immunoblotting (lower panel).

CC BY-NC-SA

no

2022-01-13 07:19:32

J Cell Biol. 2001 Sep 3; 154(5):913-924

PMC1255922

11535615

0104099f6

Figure 6. UCN-01 inhibits replication checkpoint controlling replicon firing. (A) Asynchronous CHOC cells were pulse labeled with CldU and then treated with aphidicolin for 12 h either in the absence (extreme left-hand panels, +Aphidicolin) or in the presence of 300 nM UCN-01. At time points after aphidicolin addition (0.5, 3, 6, 9, 12 h), aliquots of cells were washed free of drugs, pulse labeled with IdU and then fixed and stained with anti-CldU (green) and anti-IdU (red). Images show typical nuclei observed at indicated time points in cells that were prelabeled early, middle, or late in S phase. Blank panels indicate time points at which IdU incorporation into cells with the indicated CldU pattern were no longer detected, as these cells have progressed into G2-phase. (B–E) CHOC cells were synchronized at the G1/S boundary, and then divided into groups treated in parallel as follows. (B) Cells were either washed free of aphidicolin (colored circles) and allowed to proceed into S phase in the complete absence of drugs, or as controls, cells were maintained throughout the experiment in aphidicolin alone (B, grey circles). (C and D) Cells were maintained in the continuous presence of aphidicolin in combination with either (C) UCN-01 or (D) K-252c. At the indicated times, all cells were washed into fresh drug-free medium, pulse labeled with CldU, and then fixed and stained with anti-CldU. The percentages of S phase (CldU-positive) nuclei showing the indicated replication pattern were scored at each time point. In addition, percentages of cells in B–D that incorporated CldU were also scored (E).

CC BY-NC-SA

no

2022-01-13 07:19:32

J Cell Biol. 2001 Sep 3; 154(5):913-924

PMC1255922

11535615

0104099f7

Figure 7. UCN-01 does not affect the timing of normal S phase progression. Three asynchronous CHOC cell cultures were pulse labeled for 10 min with CldU. Two of these were then chased +/−UCN-01. A third culture was incubated in parallel in the presence of aphidicolin and UCN-01. At indicated times after CldU labeling, aliquots of cells from each were washed free of drugs, pulse labeled with IdU for 20 min, and then fixed and stained for CldU and IdU. At each time point (minimum of 100 CldU-labeled nuclei counted), the percentage of nuclei that proceeded from early CldU-labeled replication patterns to either early, middle, or late IdU replication patterns during the chase time was scored. The percentage of cells that label with IdU decreases as progressively more CldU-labeled cells complete S phase and enter G2. Results are representative of two independent experiments.

CC BY-NC-SA

no

2022-01-13 07:19:32

J Cell Biol. 2001 Sep 3; 154(5):913-924

PMC1255929

12707307

200211127f1

Figure 1. Colocalization of origins used in two consecutive cell cycles. (A) Cells were synchronized at the G1/S border with aphidicolin, released for 10 min, and then pulse labeled with CldU for 10–60 min. Labeled cells were then synchronized in mitosis and blocked at the following G1/S. Aliquots were then released for 10 min and pulse labeled with IdU for 30 min and chased for 6–13 h. DNA fibers were prepared, and CldU and IdU were detected by indirect immunofluorescence (In vivo/In vivo). In parallel, aliquots were released for 5–30 min, and nuclei from these cells were introduced into Xenopus egg extracts containing biotin-16–dUTP. In vitro replication reactions were terminated at 30 min, and DNA fibers were stained for CldU and biotin (In vivo/In vitro). (B) Exemplary fibers for each of the methods described in A are shown. In these experiments, 1–5-μm gaps between similar length (1:1 to 1:2) stretches of label were considered to be origins. Based on the lengths of purified lambda DNA molecules, we estimate fibers to be ∼2.6 kb/μm, which is in agreement with measurements made by others ( Jackson and Pombo, 1998 ). Using this estimate, a 10-min in vivo pulse label highlights 10–20-kb stretches of DNA, in agreement with the estimates of replication fork rates in mammalian cells (30 nt/s [ Brown et al., 1987 ]). (C) Coincidence of origins labeled in two consecutive in vivo cell cycles (In vivo/In vivo). The distances between origins were measured as described in Materials and methods. Only double labeled (IdU and CldU) fibers were scored (note that most fibers were singly labeled). Origins were scored as coincident if the center of the gaps for CldU and IdU were within 2 μm (∼5.2 kb) of each other. Shown are the percentage of CldU origins that were coincident with IdU origins (percentage of time that origins used in the first cell cycle were used in the second cell cycle) and the percentage of IdU origins that were coincident with CldU origins (percentage of time that origins used in the second cell cycle were used in the first cell cycle). Results of three independent experiments are shown (CldU coincident with IdU was scored in two of those three experiments) expressed as either the total data from all fibers in all three experiments or as the average coincidence for the three experiments and the variation between those experiments.

CC BY-NC-SA

no

2022-01-13 07:22:09

J Cell Biol. 2003 Apr 28; 161(2):257-266

PMC1255929

12707307

200211127f2

Figure 2. Colocalization of origins selected for initiation in vitro with those selected in vivo. (A) CHOC 400 cells were synchronized at the G1/S border with aphidicolin and then released from the aphidicolin block for 10 min. Cells were then pulse labeled with BrdU for 20–30 min and synchronized in mitosis. At 1 (pre-TDP), 2 (post-TDP), or 5.5 (post-ODP) h after mitosis, cells were collected, and intact nuclei were introduced into Xenopus egg extract supplemented with biotin-dUTP. After 30 or 40 min, nuclei were transferred to fresh extract without biotin and further incubated for 1.5–2 h. DNA fibers were then stained for BrdU and biotin. Exemplary DNA fibers are shown that display either partial overlap between BrdU and biotin label that was not scored as coincident (B) or overlap that was scored as coincident (C; many fibers did not show any overlap). In C, the green arrowheads indicate BrdU-labeled origins, and red arrowheads indicate biotin-labeled origins. Yellow arrowheads indicate the overlap. Fibers do not always show yellow label. This may be due to the interference of antibody and avidin labeling on the same DNA sites or to recurrent binding of DNA fibers to the glass surface, which interferes locally with DNA denaturation and restricts the accessibility to epitopes ( Pasero et al., 2002 ). (D) The percentage of BrdU origins that were coincident with biotin origins (percentage of time that origins used in vivo were used in vitro) and the percentage of biotin origins that were coincident with BrdU origins (percentage of time that origins used in vitro were used in vivo) was scored as in the legend to Fig. 1 . Shown are the results of four independent experiments where data were collected independently by two different investigators in a double blind manner (coincidence of biotin with BrdU was only scored in three of those experiments). Data are presented as in the legend to Fig. 1 except that p-values are included to demonstrate the significance of the differences between results obtained with pre-ODP and post-ODP nuclei. Note that the increase in biotin origins on double labeled fibers is interpreted to reflect a clustering of origins at the TDP as shown in Fig. 5 .

CC BY-NC-SA

no

2022-01-13 07:22:09

J Cell Biol. 2003 Apr 28; 161(2):257-266

PMC1255929

12707307

200211127f3

Figure 3. Interorigin distances in vitro. (A) Intact nuclei from cells synchronized at 1, 2, or 5.5 h after mitosis were introduced into Xenopus egg extract supplemented with biotin-dUTP for 30–40 min. Digoxigenin-dUTP was then added, and reactions were incubated for an additional 2 h. (B) Shown are three exemplary DNA fibers and a schematic diagram of how the interorigin distances were measured. (C) Images of DNA fibers were collected with a confocal microscope as indicated in Materials and methods, and both the lengths of the biotin labeled tracks, the number of tracks per fiber (on fibers containing more than one track), and the interorigin distances were scored. The differences in track lengths between 30 and 40 min are not generally statistically significant but are consistently longer for the 40-min labeling times, and the differences are consistent with prior measurements of the fork elongation rates with mammalian nuclei in Xenopus egg extracts ( Dimitrova and Gilbert, 1998 ). (D) Same as C except nuclei were incubated in Xenopus egg extracts either in the presence (+) or absence (−) of a nondegradable derivative of geminin to inhibit pre-RC assembly in vitro. Biotin-dUTP labeling time was 40 min. For C and D, similar results were obtained in two independent experiments.

CC BY-NC-SA

no

2022-01-13 07:22:09

J Cell Biol. 2003 Apr 28; 161(2):257-266

PMC1255929

12707307

200211127f4

Figure 4. Interorigin distances in vivo. Cells were synchronized at the G1/S border with aphidicolin, and the earliest replicating origins were labeled with IdU as in the legends to Figs. 1 – 2 . After a 30-min IdU labeling period, medium was changed to medium containing CldU, and cells were incubated an additional 1 h. To allow DNA fibers to complete replication, cells were allowed to proceed into the next G1 phase. DNA fibers were then stretched and stained for IdU and CldU as in the legend to Fig. 1 . (B) Interorigin distances were measured as in the legend to Fig. 3 . Shown are the average results from two independent experiments. (C) Histograms comparing the interorigin distances from all fibers analyzed in Figs. 3 and 4 , grouping distances into bins of 15 kb starting from 5–20 kb. (D) Relationship of interorigin distances and total fiber lengths for each dataset. When all fibers longer than the longest fiber in the in vivo dataset (dataset with the shortest fibers) are removed from each dataset, interorigin distances come into even closer agreement than for the unadjusted dataset. (E) Data for all fibers was pooled and plotted in histograms that include all fibers, only fibers longer than 180 kb, or only fibers longer than 260 kb. Also shown are the means, SD, and total number of fibers (N) for each sampling.

CC BY-NC-SA

no

2022-01-13 07:22:09

J Cell Biol. 2003 Apr 28; 161(2):257-266

PMC1255929

12707307

200211127f5

Figure 5. A similar number of origins localize to fewer DNA fibers at the TDP. (A) Using the fiber preparations collected in Fig. 3 , the percentage of DNA fibers displaying only one versus more than one origin was determined. Results indicate a significant increase in the number of fibers containing two or more biotin tracks at the TDP, with no further increase at the ODP. (B) Using the fiber preparations collected in Fig. 2 , the total number of biotin- and BrdU-labeled origins was scored for several fields of vision. For these same fields of vision, the number of fibers containing only BrdU tracks versus those containing both BrdU and biotin tracks was scored. Since preparations representing all three time points were derived from the same population of BrdU-labeled cells, these results indicate that a similar number of origins were used but that more origins were localized to early replicating DNA fibers after the TDP, with no further change at the ODP. (C) Aliquots of nuclei used for the experiments in Fig. 2 , isolated from cells at 1 (squares), 2 (circles) or 5.5 (diamonds) h after mitosis, were introduced into Xenopus egg extracts supplemented with [α- 32 P]dATP, and the percentage of input DNA replicated was determined at the indicated times by acid precipitation (e.g., 100% DNA synthesis would indicate replication of all genomic DNA). (D) In parallel, the same preparations of nuclei were incubated in extracts supplemented with biotin-dUTP, and the percentage of biotin-labeled nuclei was scored at the indicated time points. Given that the mean replication fork elongation rate is the same for each preparation (i.e., similar labeled track lengths; Fig. 3 ), the data in C and D provide independent support that the same number of replication origins are used with nuclei isolated at all three G1 phase time points.

CC BY-NC-SA

no

2022-01-13 07:22:09

J Cell Biol. 2003 Apr 28; 161(2):257-266

PMC1255929

12707307

200211127f6

Figure 6. Model for the progressive restriction of initiation potential during G1 phase. In early G1 phase, many sites distributed throughout the genome have an equal potential to be used as early replication origins. At the TDP, late replicating chromosomal domains become excluded from the pool of potential early replicating origins. At this time, origins within these early replicating domains still have an equal potential for initiation regardless of their position within the domain. At the ODP, a subset of these potential origins are chosen for initiation in the upcoming S phase.

CC BY-NC-SA

no

2022-01-13 07:22:09

J Cell Biol. 2003 Apr 28; 161(2):257-266

PMC1282461

15738051

200409187f1

F igure 1. Sequence alignment of the C-linker regions between CNGA1 and HCN2 and structural model of the C-linker regions of CNGA1. (A) Sequence alignment of the C-linker regions between CNGA1 and HCN2. Green indicates conserved sequence and yellow indicates identical sequence. The square indicates the cysteine mutation region. (B) Structural model of CNGA1 C-linker based on the crystal structure of HCN2 channel COOH termini, with red and blue used to show alternate subunits. The model was built using the software Swiss-PDB viewer in conjunction with the Swiss-Model protein modeling server ( Guex and Peitsch, 1997 ). The yellow residues indicate 420H in each subunit. The distance between 420H is 35.12 Å for adjacent subunits, and 50.18 Å for opposite subunits.

CC BY-NC-SA

no

2022-01-13 09:51:40

J Gen Physiol. 2005 Mar; 125(3):335-344

PMC1282461

15738051

200409187f2

F igure 2. Cu/P induced potentiation in three of the seven cysteine mutants. Dose–response curves of cysteine mutants and CNGA1 cysless activated by cGMP initially (open circles) and after (filled circles) treatment with Cu/P plus 2 mM cGMP. Smooth curves represent fits with the Hill equation (see materials and methods ), with mean K 1/2 values listed in Table I .

CC BY-NC-SA

no

2022-01-13 09:51:40

J Gen Physiol. 2005 Mar; 125(3):335-344

PMC1282461

15738051

200409187f3

F igure 3. Cu/P effect on the current activated by saturating cAMP. Fraction of the current activated by 20 mM cAMP versus 2 mM cGMP (IcAMP/Imax) initially (open circles) and after Cu/P treatment (filled circles). Points represent the mean, with error bars representing the SEM. Patch numbers range from 4 to 8. Red * denotes a statistical difference of P < 0.01 (Student's t test) between the initial data and that after Cu/P treatment.

CC BY-NC-SA

no

2022-01-13 09:51:40

J Gen Physiol. 2005 Mar; 125(3):335-344

PMC1282461

15738051

200409187f4

F igure 4. DTT reversed the effect of Cu/P treatment. (A) DTT reversed Cu/P potentiation of 417C. cGMP dose–response curves initially (open circles), after a 5-min treatment with Cu/P plus 128 μM cGMP (black circles), and after treatment with 5 mM DTT + 64 μM cGMP for 10 min (red circles). The smooth curves are fits with the Hill equation, with the following parameters: initially K 1/2 = 7.04 μM, Hill slope = 2.0 (black curve, right), after Cu/P K 1/2 = 1.61 μM, Hill slope = 2.8 (black curve, left), after DTT K 1/2 = 5.03 μM, Hill slope = 2.2 (red curve). (B) Time course of DTT reversing the potentiation effect of Cu/P on 417C. Normalized currents activated by 2 μM cGMP (black circles) and 2 mM cGMP (open circles) at +100 mV after a 5-min treatment with Cu/P plus 128 μM cGMP, and then various cumulative time in 5 mM DTT plus 64 μM cGMP. Smooth curve represents single exponential fit of the data, with time constant = 37.0 s.

CC BY-NC-SA

no

2022-01-13 09:51:40

J Gen Physiol. 2005 Mar; 125(3):335-344

PMC1282461

15738051

200409187f5

F igure 5. Cu/P produced two populations of channels in 418C. Single channel currents measured at +50 mV were recorded before and after a 10-min treatment with Cu/P plus 2 mM cGMP for 418C (A and B) and CNGA1 cysless channels (C). For 418C, 4 μM cGMP was used to activate the channels, and for CNGA1 cysless , 2 μM cGMP was used to activate the channels. All-points histograms were made from the data collected under each condition (right, in all cases >5 s of data was used).

CC BY-NC-SA

no

2022-01-13 09:51:40

J Gen Physiol. 2005 Mar; 125(3):335-344

PMC1282461

15738051

200409187f6

F igure 6. Mechanisms of the dual effects of Cu/P on 418C. (A) Disulfide bonds formed between opposite and adjacent subunits could produce different effects. (B) The number of disulfide bonds could determine whether potentiation or inhibition would be observed. (C and D) Kinetics of the Cu/P effect on 418C with currents activated by either (C) 2 μM cGMP or (D) 2 mM cGMP. The smooth curves represent Gaussian fits (C) and exponential fits (D).

CC BY-NC-SA

no

2022-01-13 09:51:40

J Gen Physiol. 2005 Mar; 125(3):335-344

PMC1282461

15738051

200409187f7

F igure 7. Cartoon model of closed and open channels. Side (top) and top (bottom) views of the CNGA1 C-linker made from the HCN2 structure (A) and from a model based on our data (B). The A' helices are shown in green and the B' helices are shown in blue. A and B differ only by the conformation of the B'-C' loop.

CC BY-NC-SA

no

2022-01-13 09:51:40

J Gen Physiol. 2005 Mar; 125(3):335-344

PMC1283094

16103225

200504131f1

Figure 1. Targeting of the Sgk3 gene by homologous recombination. (A) Schematic of WT mouse Sgk3 domain structure, gene locus, targeting construct, and mutated Sgk3 allele. Exons 8 and 9 were replaced with PGKneo, and diphtheria toxin A was used for negative selection. Indicated are the antibody binding site, the 5′ external probe A that was used for Southern hybridization, predicted sizes of hybridizing fragments, and primer pairs that were used for PCR. Short arrows represent the primers that were used for PCR analysis of WT (a and b) or mutant (c and d) alleles. RV, EcoRV; Xb, XbaI; S, SmaI. (B) Southern hybridization analysis (SB) of F2 offspring of intercrosses of Sgk3 mutant F1 mice. Tail genomic DNA digested with XbaI was probed with external probe A; W, WT allele; M, mutant allele. (C) Northern analysis (NB) of Sgk3 mRNA expression in total mRNA extracted from P5 skin. EtBr, ethidium bromide. (D) Western immunoblot (WB) shows the presence of Sgk3 in cultured keratinocytes. (E) Immunoblot of dispase-separated epidermis and dermis hair follicle (HF) fractions shows Sgk3 (arrows) in the hair follicle fraction but not in the interfollicular epidermal fraction and also demonstrates that Sgk3 is expressed throughout the hair cycle in whole skin. Extra bands on Western blots are a result of nonspecific signal. Actin, loading control.

CC BY-NC-SA

no

2022-01-13 07:26:07

J Cell Biol. 2005 Aug 15; 170(4):559-570

PMC1283094

16103225

200504131f2

Figure 2. Sgk3 is required for normal hair coat production. Adult mice (P36 and P411) have a sparse, uneven hair coat (A–D) and irregular hair shafts when seen in profile (E and F). Whiskers are malformed (G and H). Plucked hairs (I and J) lack normal guard, awl, or zigzag hairs. Scanning EM (K and L) shows KO hairs to be irregular and thin with occasional malformed cuticles (arrowheads). Sgk3-null plucked hairs are shorter than WT hairs, with the exception of guard hairs (M). Histology of adult back skin demonstrates that a sparse coat is not caused by the loss of hair follicles (arrows; N–Q). Transmission EM (TEM) of P3 follicles verifies the presence of all cell types in KO follicles (R and S), including the three layers of the IRS. Note the normal keratinization of Henley's layer of the IRS in the upper bulb (asterisks). HS, hair shaft; Ci, IRS cuticle; Hu, Huxley's layer of the IRS; He, Henley's layer of the IRS; ORS, outer root sheath.

CC BY-NC-SA

no

2022-01-13 07:26:07

J Cell Biol. 2005 Aug 15; 170(4):559-570

PMC1283094

16103225

200504131f3

Figure 3. Hair follicle morphogenesis is normal in Sgk3-null mice until P2. At P2, follicles have normal histology by hematoxylin and eosin (A and B), including ORS, IRS, and hair shaft (HS) layers. Follicle number and density is normal (C and D). Differentiation markers AE13 (hair keratins; hair shaft cortex), AE15 (trichohyalin; IRS), and keratin 6 (K6; companion cell layer [CCL]) are normal in KO follicles at P2 (E–H). Wnt pathway member Lef1 (lymphoid enhancer factor 1) is normal (E and F); mating Sgk3-null mice to Wnt reporter TOPgal mice reveals normal intensity of reporter activity in KO hair follicles at P2 and P6 (I–L). Arrows represent blue Xgal precipitate, demonstrating activity of the TOPgal Wnt reporter. Immunoblot of whole skin lysates at P2 confirms equal amounts of β-galactosidase (M). Mx, matrix; DP, dermal papilla; Epi, epidermis; De, dermis; Pc, precortex; Mg, melanin granules. DAPI, nuclear counterstain; Xgal, β-galactosidase enzymatic substrate. Bars (A, B, and E–H), 20 μM; (C, D, and I–L) 100 μM.

CC BY-NC-SA

no

2022-01-13 07:26:07

J Cell Biol. 2005 Aug 15; 170(4):559-570

PMC1283094

16103225

200504131f4

Figure 4. Anagen maturation is impaired in Sgk3-null hair follicles. Histology is similar at P2 (A and B) but by P6, Sgk3-null follicles are smaller and fail to grow down to the bottom of the subcutis (C and D). At P6, the shortened KO bulb has a smaller, rounded DP relative to the long, thin DP of WT follicles (G and H). Apoptotic cells are seen in some KO follicle bulbs (J and K); although many P6 bulbs show no apoptotic cells, some have as many as six. Incorporation of BrdU in KO bulbs at P6 is reduced relative to WT bulbs (L–O). Arrows represent examples of BrdU-positive nuclei. (I) Quantification shows that total cell number is reduced in KO bulbs at P6, apoptotic cell number is increased, and proliferation is markedly decreased. Epi, epidermis; De, dermis; SubQ, subcutis; Mx, matrix; Mg, melanin granules; TEM, transmission EM; AP, alkaline phosphatase. Bars (A–D), 100 μM; (E–H and L–O) 20 μM; (J and K) 10 μM.

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Figure 5. BrdU fate mapping distinguishes between models for reduced bulb cell number and proliferation. (A) Experimental design; mice pulsed with BrdU at P4 were chased for 12 or 20 h. (B) Predicted outcomes for three models. The percentage of Brdu+ cells reflects the percentage of total bulb cells that are BrdU+. The No. of BrdU+/Diff+/bulb reflects the cells per bulb that are positive for BrdU and are above the boundary (dotted lines) of differentiation marker expression. The percentage pf BrdU+/Diff+ is the percentage of BrdU+ cells that are above the boundary relative to the total. Distinguishing between an intrinsic proliferative defect in matrix cells (models 1 and 2) and a defect in the recruitment or regeneration of matrix cells (model 3) relies on the measurement of the percentage of BrdU+ cells. (C) Representative sections demonstrate movement of BrdU-labeled cells. Note that the total number of BrdU-positive cells at 20 h is double the number at 4 h, which is consistent with the expected division after S phase. This confirms that ongoing labeling beyond 4 h does not occur. Dotted lines mark the lower limit of hair keratin expression. HS, hair shaft; Mx, matrix. Bars, 20 μm. (D) Quantification reveals that the most likely explanation for reduced cell number is impaired recruitment or regeneration of progenitors. Error bars represent SEM.

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Figure 6. Hair follicles lacking Sgk3 enter catagen prematurely. Histology shows the onset of catagen in WT skin at P15 and in KO skin at P9 (A–J). (EE) Quantification of hair cycle stage based on DP morphology that was visualized by alkaline phosphatase ( Muller-Rover et al., 2001 ). Anagen (alI stages); early catagen (catagen I–III); late catagen (IV–VIII). Note that Sgk3-null follicles enter catagen prematurely; by P15, they have entered the second anagen. (K–T) BrdU staining and DP morphology suggest that KO catagen traverses normal stages. Arrows represent BrdU-positive nuclei. (O) Straight dotted line demarcates the bottom of the permanent portion of the hair follicle. (FF) Quantification of BrdU incorporation confirms the appropriate decline in proliferation as KO follicles enter catagen. (U–DD) Staining for activated caspase 3 shows that apoptosis in Sgk3-null follicles increases normally during catagen, excluding massive apoptosis as a mechanism to explain reduced bulb cell number or premature catagen. (Y) Plus signs indicate autofluorescence of the hair shaft (+) and differentiated epidermis (++). Arrowheads, caspase (+) cells. (GG) Quantification of apoptosis confirms the increased number of apoptotic cells in KO skin. Mx, matrix; Ana, anagen; Cat, catagen; Mg, melanin granules; DP, dermal papilla; Cb, Club hair. Error bars represent SEM. Bars (A–J), 100 μM; (K–DD) 20 μM.

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J Cell Biol. 2005 Aug 15; 170(4):559-570

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Figure 7. Growth factor signaling elements are present in the hair follicle. (A) Schematic depicting EGF and IGF-1 pathways and known effects on the anagen to catagen transition. Note the strong evidence that EGF and EGFR promote catagen, which are consistent with the observation that Pten, which is inhibitory to PI3K, may promote anagen. (B–I) Localization of growth factor signaling in the hair follicle. Phospho-Akt is present strongly in the upper ORS, colocalizing with K5, in both WT and KO skin at P6 (B–E). EGFR is expressed in the ORS, colocalizing with K5, and in the interfollicular epidermis and hair bulb (F–I). Arrowheads indicate colocalization with K5 in the ORS.

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J Cell Biol. 2005 Aug 15; 170(4):559-570

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Figure 8. Growth factor signaling is abnormal in Sgk3-null keratinocytes. (A) Treating WT and KO primary keratinocytes with 10 ng/ml EGF reveals normal kinetics of the activation of ERK 1/2 and Akt. (B) In response to 50 ng/ml IGF-1, Sgk3-null keratinocytes exhibit increased activation of ERK 1/2 and Akt. (C) In primary keratinocytes, IGF-1–mediated activation of ERK 1/2 is dependent on PI3K activation, whereas EGF-mediated ERK activation proceeds independently of PI3K. Cells were pretreated with 100 nM wortmannin for 30 min before stimulation. (D) PI3K-dependent IGF-1 activation of ERK does not indicate codependence of PI3K and ERK pathways; phosphorylation of Akt is unaffected by treatment with MEK inhibitor 444937. Cells were pretreated with 1 μM 444937 for 30 min before stimulation. Antibodies are against phosphorylated (activated) or total ERK 1/2 or AKT 1–3 proteins.

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J Cell Biol. 2005 Aug 15; 170(4):559-570

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200409228f1

F igure 1. ADP shortens the open time of ΔR/D1370N-CFTR channels. (A) Single-channel current traces in the presence of 1 mM ATP, 1 mM ATP + 1 mM ADP, or 1 mM ATP again. (B) Effects of ADP on the mean open and closed times. Notice that ADP shortens the mean open time and increases the mean closed time ( n = 5). Error bars represent SEM. * indicates P < 0.01.

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F igure 2. Macroscopic current relaxation for ΔR/E1371S-CFTR channels in the presence of ATP and ADP. (A) A sample trace of current relaxations for ΔR/E1371S-CFTR channels opened with 1 mM ATP, and subsequently with 1 mM ATP + 2 mM ADP. (B) The current decay upon removal of ATP can be fitted with a single exponential function with a time constant of 100.00 ± 0.02 s. (C) The current decay upon removal of ATP plus ADP is fitted with a double exponential function with time constants of 12.9 ± 0.1 and 105 ± 3 s.

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F igure 3. Macroscopic current relaxation for ΔR/E1371S-CFTR opened with 10 μM ATP. ΔR/E1371S-CFTR channels were activated with 10 μM ATP until the current reached a steady state. Then the nucleotide was washed out. (A) Sample trace. (B) Ensemble currents were generated by pooling data from 22 experiments. The insets show the first two components of the current relaxation. The dash line represents current relaxation of ΔR/E1371S-CFTR upon removal of 1 mM ATP (from Fig. 2 B).

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F igure 4. Single-channel recording of ΔR/E1371S-CFTR in the presence of 1 μM ATP. A continuous, 54-min single-channel trace in the presence of 1 mM ATP. Note that the channel is open most of the time. The Po is almost 1.

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F igure 5. Single-channel recording of ΔR/E1371S-CFTR in the presence of 10 μM ATP. (A) A continuous 45-min single-channel trace in the presence of 10 μM ATP. Note that the channel remains closed for long periods, and presents opening bursts of different lengths. (B) Expanded traces of selected parts of the trace in A. Note the presence of very brief openings (*), and intermediate locked-open events (**), and rarely occurring long locked-open events (***).

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F igure 6. Single-channel dwell time analysis of ΔR/E1371S-CFTR. (A) Events from two single-channel recordings (∼50 min each) were pooled together to construct this closed time histogram. The closed time distribution for data obtained at 10 μM ATP can be fitted with a quadruple exponential function. No cutoff was used for the construction of this histogram. The arrow marks the cutoff (500 ms) used to define the minimum of the ATP-dependent closed times used for the open time analysis in D. (B) The closed time distribution for data obtained at 3 μM ATP and fitting parameters. (C) Events from two recordings of single channels in the presence of 1 mM ATP were pooled together to construct this closed time histogram (∼100 min of recording). The closed time distribution can be fitted with a triple exponential function. No cutoff was used for the construction of this histogram. The arrow marks the cutoff used to construct the open time histogram shown in F. (D) The open time histogram for data obtained at 10 μM ATP shows the presence of three distinct burst lengths: a brief opening of 330 ms, an intermediate burst of 6 s (most of the events), and very few lock-open bursts with a time constant of ∼100 s. (E) The open time histogram for the recording at 3 μM ATP. (F) The open time histogram shows that most of the events are long-lived locked-open bursts (>50%).

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F igure 7. Intraburst kinetic analysis. Locked-open bursts were divided into two categories: short-lived locked-open events (1–50 s in length) and long-lived locked-open events (>50 s). Both types of bursts show similar characteristics. The closed dwell time histograms (A and C) show two components: the flickers (20–30 ms) and a relatively longer closing (∼100 ms). Once the flickering closures are removed by using a 50-ms cutoff, the open times within the locked-open bursts are very similar, irrespective of the locked-open duration (B and D).

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F igure 8. Macroscopic current relaxation of E1371S-CFTR currents. (A) Sample trace of current relaxations for E1371S-CFTR channels activated with 10 μM ATP + PKA, or with 1 mM ATP + PKA. (B) Normalized ensemble current relaxations upon removal of 1 mM ATP (from 12 patches) or 10 μM ATP (from 9 patches). The 1 mM ATP relaxation curve can be fitted with a double exponential function (red curve) with time constants of 149.3 ± 0.02 s (69%) and 29.59 ± 0.02 s (31%). The 10 μM ATP curve can be fitted also with a double exponential function (blue curve) with time constants of 107.53 ± 0.08 s (29%), and 26.32 ± 0.01 s (71%).

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F igure 9. Kinetic analysis of the last E1371S-CFTR channel that remains open after removal of ATP. (A) Sample trace of the current relaxation of E1371S-CFTR channels upon ATP washout. The inset shows the expanded trace of the last channel that remains open. Note the presence of poorly resolved flickers and several long closings that last for hundreds of milliseconds. (B) Data from four patches were pooled together to construct the closed time histogram.

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F igure 10. The K464A mutation shortens the locked-open time of E1371S-CFTR. (A) Sample trace of K464A/E1371S-CFTR channels in the presence of 1 mM ATP + PKA. Note that the relaxation upon nucleotide washout is very fast. (B) Ensemble macroscopic currents were generated from 18 patches. The macroscopic current has a relaxation time constant of 19.60 ± 0.01 s (red curve). (C) Sample trace of K464A/E1371S-CFTR channels in the presence of 10 μM ATP (blue curve). The inset shows the presence of numerous brief openings (from 42 to 650 ms). Note also the presence of longer openings (*, 9 s).

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F igure 11. Kinetic analysis of the spontaneous openings in the absence of ATP. (A) A representative ΔR/E1371S-CFTR current trace from an excised inside-out patch exposed to ATP-free solution for several minutes before 1 mM ATP was applied. (B) Similar experiment as described in A, but with ΔR-CFTR channels. The open times of these spontaneous openings from several patches containing ΔR/E1371S-CFTR (C) or ΔR-CFTR (D) were pooled together to construct survivor plots.

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J Gen Physiol. 2005 Apr; 125(4):377-394

PMC1307503

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0110059f1

Figure 1. I SOCE during Xenopus oocyte maturation. (A) Voltage protocol used to measure I SOCE development and I–V relationship. The membrane potential was stepped to −140 mV (100 ms), and then ramped from −140 to +60 mV (2 s) from a −40 mV holding potential. The protocol was applied once every 30 s. (B and C) I SOCE development after Ca 2+ store depletion with ionomycin (10 μM) in an oocyte (B) and an egg (C). Cells were injected with 7 nmol BAPTA to allow for I SOCE recording ( Machaca and Haun, 2000 ). The current at −140 mV (A, arrow) was plotted over time. La 3+ (100 μM) was added at the end of the experiment to block SOCE and obtain a measure of absolute I SOCE . (D) Representative I–V relationships obtained from the ramp voltage stimulation shown in A from an oocyte and an egg. I–V relationships were obtained by subtracting the current after La 3+ addition from the current before addition. (E) Time course of progesterone-mediated GVBD. Progesterone was added to a population of at least 50 oocytes, and the occurrence of GVBD over time was recorded by following white spot appearance on the animal pole. The data shown represent the average ± SE from seven donor females. The time of GVBD i ± SD is also shown. GVBD i represents the time at which GVBD was first observed in the population. (F and G) I SOCE levels from individual oocytes at different times during oocyte maturation. Each square represents the current from a single cell. I SOCE data from oocytes are shown before progesterone addition. The data was normalized to average current in oocytes. I SOCE data from 80 cells at different times during maturation are shown in F. (G) I SOCE levels from cells incubated in progesterone without undergoing GVBD ( n = 63). The average time ± SD of GVBD i is shown for reference.

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J Cell Biol. 2002 Jan 7; 156(1):75-86

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Figure 2. Time course of MPF, MAPK, and Mos activation during progesterone-induced maturation. (A) Time course of GVBD. GVBD i occurred at 5.5 h, and GVBD 50 ∼7.25 h after progesterone addition. At each time point, five cells were pooled to quantify kinase activity. (B–D) MPF, P-MAPK, and Mos levels during oocyte maturation. MPF activity was measured as the histone H1 kinase activity as described in Materials and methods. The levels of P-MAPK and Mos were determined by Western blot analysis using a P-MAPK–specific and anti-Mos antibody, respectively, as described in Materials and methods. In the case of MAPK, the same blot was reprobed with an antibody that detects total MAPK (T-MAPK) levels, showing that MAPK was present in the lysates but was not phosphorylated. To be able to compare P-MAPK and Mos levels between different experiments, we always ran a positive control lysate from eggs on every gel, and normalized P-MAPK and Mos levels to that sample. Therefore, Western data for P-MAPK and Mos throughout this manuscript are normalized to the same egg lysate (see Materials and methods for more details). The time scale is divided into two phases: after progesterone addition; and after GVBD. The 35-kD molecular mass marker is shown on the right of the gels. (E) MPF, P-MAPK, and Mos levels in cells treated with progesterone, but that did not undergo GVBD. GVBD i is indicated for reference. This time course of kinase activation is representative of five similar experiments.

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Figure 3. Correlation between I SOCE and MPF and P-MAPK levels after Δ87cyclin B1 protein injection. Oocytes were either directly injected with Δ87cyclin B1 protein (A and B) or preincubated in the MEK inhibitor PD98059 (50–100 μM) for 0.5–2 h before Δ87cyclin B1 injection. I SOCE levels were recorded at various times after Δ87cyclin B1 injection. After I SOCE recording, individual cells were lysed and assayed for MPF and P-MAPK levels. This allowed us to obtain I SOCE (squares), MPF (circles), and P-MAPK (triangles) levels from the same oocyte. The data for individual cells are connected by drop lines to help in matching I SOCE and kinase activity from the same cell. For Δ87cyclin B1 and PD98059-Δ87cyclin B1 injections, current and kinase data from 40 and 33 cells, respectively, are shown. I SOCE levels were normalized to the levels in untreated oocytes, and MPF and MAPK levels were normalized to the levels found in fully mature eggs, as described in Fig. 2 . The time scale was normalized to the time at which GVBD initially appeared in the population (GVBD i ) after Δ87cyclin B1 injection. The standard deviation of GVBD i is shown in A and B ( n = 7). PD98059-treated cells are plotted on a time scale normalized to GVBD i in Δ87cyclin B1–injected cells (A and B). Individual cell data were fitted with a Boltzman function to obtain a general trend of I SOCE inactivation and the activation of the different kinases.

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Figure 4. Representative individual cell data from Δ87cyclin B1–injected cells. MPF, P-MAPK, and Mos Western data are shown in conjunction with the levels of each kinase in fully mature eggs (EGG). (A) I SOCE was activated in response to store depletion with ionomycin (10 μM) in an oocyte, and MPF, P-MAPK, and Mos were not detected. (B) A representative oocyte injected with Δ87cyclin B1. I SOCE was not activated in response to store depletion. MPF and P-MAPK levels were 1.6 and 0.9 times those in eggs, whereas Mos kinase was not detected in this oocyte. (C) A representative oocyte preincubated with PD98059 (100 μM) before Δ87cyclin B1 injection. I SOCE was not activated in response to store depletion, MPF levels were 2.7 times higher than those found in the control egg lysate, and both P-MAPK and Mos were not detectable.

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J Cell Biol. 2002 Jan 7; 156(1):75-86

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Figure 5. Correlation between I SOCE and Mos, P-MAPK, and MPF levels after injection of GST–Mos RNA. GST–Mos is a fusion protein between GST and Xenopus Mos, which allowed the separation between endogenous Mos (39 kD) and recombinant, injected GST–Mos (64 kD) (see Materials and methods). Oocytes were either directly injected with 10 ng GST–Mos RNA (A–C) or preinjected with 10 ng of GST–107Wee1 (Wee) 13–16 h before Mos RNA injection. GST–107Wee1 is a constitutively active form of the Wee1 kinase, which blocks MPF activation ( Howard et al., 1999 ). I SOCE levels recorded at various times after GST–Mos RNA injection. After I SOCE recording, individual cells were lysed and assayed for kinase activities. This allowed us to obtain I SOCE (squares), Mos (diamonds), P-MAPK (triangles), and MPF (circles) levels from the same oocyte. For Mos and Wee1–Mos injections, current and kinase data from 40 and 27 cells, respectively, are shown. The plotted Mos data is for GST–Mos protein levels, as endogenous Mos was not detected in these experiments. The data for individual cells are connected by drop lines to help in matching I SOCE and kinase activity from the same cell. I SOCE levels were normalized to the levels in untreated oocytes, and Mos, MAPK, and MPF levels were normalized to the levels found in fully mature eggs as described in Fig. 2 and Materials and methods. The time scale was normalized to GVBD i , the standard deviation of which is shown in A–C ( n = 5). Cells preinjected with GST–107Wee1 were plotted on the same time scale as GST–Mos-injected cells (D–F). Individual cell data were fitted with a Boltzman function to obtain a general trend of I SOCE inactivation and the activation of the different kinases.

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Figure 6. Representative individual cell data from Wee1- and Mos-injected cells. MPF, P-MAPK, and Mos Western data are shown in conjunction with the levels of each kinase from a lysate of fully mature eggs (EGG). (A) I SOCE was activated in response to store depletion with ionomycin (10 μM) in an oocyte. MPF, P-MAPK, and Mos were not detected in this cell. (B) A representative cell injected with 10 ng GST–Mos RNA. I SOCE was not activated in response to store depletion. I SOCE was recorded at the 1GVBD i time point in this cell, i.e., at about the same time that GVBD first occurred in the population. MPF and P-MAPK levels were 1.3 and 1.6 times, respectively, higher than those in the egg lysate. GST–Mos protein (arrowhead) levels were 13.1 times higher than egg levels and no endogenous Mos protein (arrow) was detected. (C) A representative cell preinjected with 10 ng GST–107Wee1 RNA 14 h before GST–Mos RNA (10 ng) injection. We recorded I SOCE at the 1GVBD i time point. SOCE was readily activated, in response to store depletion, to levels similar to those found in oocytes. Injection of GST–107Wee1 effectively blocked MPF activation, which was detected at 0.2 times egg level. In contrast, P-MAPK and GST–Mos (arrowhead) were detected at 0.76 and 8.7 times egg levels, respectively.

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Figure 7. Continuous recording of I SOCE through the GVBD transition. (A) Prolonged recording of I SOCE from a control untreated oocyte. MPF activity in this cell is also shown. (B) I SOCE was activated in a progesterone-treated oocyte before GVBD and continuously recorded until, and past, the time point at which GVBD occurred (arrow). At the end of the experiment, the cell was lysed in extraction buffer and MPF activity was measured. This cell had high MPF activity, indicating that the prolonged recording did not interfere with MPF activation at GVBD. MPF activation at GVBD is not able to block I SOCE that has been preactivated before GVBD.

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Figure 8. Pre-activated I SOCE does not inactivate in response to MPF activation. Oocytes were incubated in Ca 2+ -free medium (L-15 with Ca 2+ , buffered at 50 μM), containing thapsigargin (1 μM) to deplete intracellular Ca 2+ store and activate I SOCE . Cells were voltage clamped in a Ca 2+ -free solution (70Mg; see Materials and methods) and then switched to a solution containing 30 mM Ca 2+ (30Ca; see Materials and methods) to induce Ca 2+ influx through SOCE channels. (A) I SOCE and MPF levels from a control thapsigargin–treated oocyte. (B) Oocytes were treated with progesterone (5 μg/ml) for 1 h and then thapsigargin (1 μM) was added, and the cells were incubated until GVBD occurred. I SOCE was measured ∼15 min after GVBD occurrence. I SOCE was still present in this cell, although MPF was activated at high levels, indicating that MPF is unable to block SOCE that has been preactivated before MPF induction. I SOCE (C) and MPF (D) levels in oocytes ( n = 5) and cells treated with progesterone 10–15 min ( n = 8) and 15–30 min after GVBD ( n = 7). I SOCE levels gradually declined after GVBD and MPF levels remained relatively stable.

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J Cell Biol. 2002 Jan 7; 156(1):75-86

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200505155f1

Figure 1. Phosphorylation of the α 1C subunit by CaMKII. (A) Autoradiogram showing phosphorylation of α 1C by CaMKII. Lysates from HEK cells transfected with α 1C and β2b (lanes 2–4) or nontransfected cells (lane 1) were immunoprecipitated with an anti-α 1C antibody (lanes 1, 3, and 4) or control IgG (lane 2) and then incubated with purified α-CaMKII in the presence of Ca 2+ /CaM and Mg 2+ /ATP 32 as described in Materials and methods. 200 nM of the CaMKII inhibitor peptide AIP-2 (Calbiochem) was included (lane 4) to demonstrate kinase specificity. Phosphorylated α 1C is indicated by an arrowhead; autophosphorylated CaMKII, retained after the kinase reaction despite extensive washing of the immunoprecipitate, is indicated with a double arrowhead. An anti-α 1C immunoblot of the samples used in the kinase reaction is shown below the autoradiogram. (B) Schematic of α 1C . Thick black lines highlight regions used to generate GST fusion proteins. (C) GST fusion proteins enriched from bacterial lysates using glutathione–sepharose were incubated with purified α-CaMKII in the presence of Ca 2+ /CaM and Mg 2+ /ATP 32 as described in Materials and methods. After extensive washes, proteins were eluted using SDS-PAGE sample buffer. Autoradiogram of fusion proteins separated by SDS-PAGE after phosphorylation by CaMKII. C-term refers to the more distal COOH-terminal fusion protein, containing aa 1669–2171. Above the autoradiogram is the Coomassie blue–stained band for each fusion protein, indicating nearly equal loading of substrate for all fusion proteins.

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J Cell Biol. 2005 Nov 7; 171(3):537-547

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Figure 2. CaMKII coimmunoprecipitates and colocalizes with α 1C . (A) Biotinylated calmodulin overlay of rat cardiac sarcolemmal membranes after immunoprecipitation with an anti-α 1C antibody. Purified α-CaMKII was run as a control to demonstrate effectiveness of CaM overlay. An anti-α 1C antibody (but not control IgG) coimmunoprecipitated a protein identified as the δ isoform of CaMKII by biotinylated CaM overlay and apparent molecular mass. (B) Anti-GFP immunoblot after immunoprecipitation of GFP-CaMKII by control IgG (lane 4) or anti-α 1C antibody (lane 5) from lysates of HEK 293 cells transiently transfected with GFP-CaMKII and α 1C .

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J Cell Biol. 2005 Nov 7; 171(3):537-547

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Figure 3. Activity-dependent interaction of CaMKII with the cytoplasmic determinants of α 1C . Immunoblots using an mAb (CBα2) for CaMKII after a GST pull-down assay with 20 nM of native (top), Ca 2+ /CaM-activated (middle), or Ca 2+ /CaM/autophosphorylated α-CaMKII (bottom). GST fusion proteins contained various cytoplasmic regions of α 1C just as in Fig. 1 C. Panel above the immunoblots shows a representative Ponceau stain of each fusion protein. Although only one Ponceau staining profile is shown, all blots were run in parallel, and equal loading of all fusions proteins was independently verified.

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J Cell Biol. 2005 Nov 7; 171(3):537-547

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Figure 4. Localization of the CaMKII binding site on the COOH terminus of α 1C . (A) Diagram of α 1C fusion proteins used in GST pull-down assays with autophosphorylated α-CaMKII, exhibiting robust (+), partial (±), and no (−) binding. (B) Immunoblot with CBα2 after GST pull down of 20 nM of purified autophosphorylated α-CaMKII, using α 1C aa 1581–1690 fused to GST. Pull-down assay performed in the presence of 40 μM of the indicated peptide or the peptide diluent DMSO. (C) Quantification after immunoblot with CBα2 of GST pull-down assays of purified autophosphorylated α-CaMKII, using α 1C aa 1581–1690 (wild type [WT]), a 1644 TVGKFY 1649 → EEDAAA mutant (Mut6), or GST alone shows that Mut6 blocks CaMKII binding. Panel above the immunoblots shows a representative Ponceau stain of each fusion protein. *, P < 0.001 for a one-way analysis of variance followed by Dunnett's test to identify specific pair-wise differences between the means for Mut6 versus WT and GST versus WT ( n = 4–8). Inset shows an exemplar immunoblot with CBα2. (D) An exemplar immunoblot with an anti-CaM antibody, showing that CaM binding is not affected by the Mut6 mutation. Panel above the immunoblots shows a representative Ponceau stain of each fusion protein.

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J Cell Biol. 2005 Nov 7; 171(3):537-547

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16275756

200505155f5

Figure 5. CaMKII interaction with the COOH terminus of α 1C is essential for CDF. (A) I Ba and scaled I Ca traces during a train of 40 test pulses of V h from –90 mV to +20 mV at 3.3 Hz recorded from oocytes expressing α 1C I1654A (I/A) or α 1C I1654A/ 1644 TVGKFY 1649 → EEDAAA (I/A-Mut6). Bars, 500 nA and 25 ms. (B) Peak I Ba and I Ca during trains of 40 repetitive test pulses at 3.3 Hz, normalized to the current amplitude at the beginning of each train ( n = 4–5). Values indicate means ± SEM. (C) Changes in peak I Ba and I Ca conducted by α 1C I1654A (I/A) or α 1C I1654A/ 1644 TVGKFY 1649 → EEDAAA (I/A-Mut6) at indicated stimulation frequencies ( n = 4–5) Values indicate means ± SEM. (D) Summary of the recovery from inactivation after a two-step protocol for I/A and I/A-Mut6. The length of the prepulse was individually determined for each oocyte to produce ∼75–90% inactivation. (E) Autoradiograph showing phosphorylation of wild type (WT) or mutant α 1C (Mut6) by CaMKII, performed as in Fig. 1 A. An anti-α 1C immunoblot of the samples used in the kinase reaction confirmed similar expression levels of the WT and mutant α 1C subunits. An anti-CaMKII immunoblot with CBα2 confirmed the identity of the retained 50-kD 32 P-labeled protein as α-CaMKII.

CC BY-NC-SA

no

2022-01-13 07:26:11

J Cell Biol. 2005 Nov 7; 171(3):537-547

PMC1343528

16275756

200505155f6

Figure 6. The binding site for the COOH terminus of α 1C on CaMKII is localized near the catalytic domain. (A) Biotinylated CaM overlay of GST pull downs, using a fusion protein from the COOH terminus of α 1C (aa 1509–1905) on lysates of HEK 293 cells transiently transfected with the CaMKII isoforms (α, β, δ A , δC, and γ B ; arrows) after thioautophosphorylation. In lanes 6 and 7, lysates of untransfected cells were run with (+) and without (−) purified thiophosphorylated α-CaMKII added to the lysate. (B) Immunoblot using an mAb (CBα2) for CaMKII after GST pull downs, using a fusion protein from the COOH terminus of α 1c (aa 1509–1905) and 20 nM of purified autophosphorylated α-CaMKII. In addition, 20 μM of the indicated peptide was added to each binding reaction. (C) Sequence alignment of CaMKII binding sites from the COOH termini of NR2B and α 1C with the autoregulatory domain from α-CaMKII.

CC BY-NC-SA

no

2022-01-13 07:26:11

J Cell Biol. 2005 Nov 7; 171(3):537-547

PMC1343528

16275756

200505155f7

Figure 7. CaMKII interaction with the COOH terminus of α 1C is not reversed by dephosphorylation or CaM dissociation, and tethered CaMKII requires autophosphorylation or Ca 2 + /CaM for activity. (A and B) Immunoblots with CBα2 or a phosphospecific CaMKII mAb after GST pull-down assays, using α 1C aa 1509–1905 and 20 nM of autophosphorylated α-CaMKII. (A) 5 mM EGTA was present in the binding reaction and/or in the wash. (B) Purified recombinant PP1 was added before (PP1-Pre) or after (PP1-Post) the binding reaction in the presence or absence of 5 mM EGTA, as indicated. (C) Time course of reversal of CaMKII autonomous activity after PP1 treatment in solution ( n = 4). (D) Activity measurements, using peptide AC-2 as a substrate, of CaMKII recovered in GST pull-down assays, using α 1C aa 1509–1905. Ca 2+ /CaM-dependent and autonomous activity measurements of CaMKII recovered after treatment with recombinant PP1 for 30 min (PP1) or no treatment (−) in the binding assay ( n = 4) Values indicate means ± SD.

CC BY-NC-SA

no

2022-01-13 07:26:11

J Cell Biol. 2005 Nov 7; 171(3):537-547

PMC1343528

16275756

200505155f8

Figure 8. Proposed mechanism of CaMKII binding to α 1C to form a local and dedicated Ca 2 + spike integrator for CDF. A catalytic core and autoregulatory domain for a prototypical CaMKII inactive subunit is shown on the bottom left (inactive is indicated by green). Ca 2+ /CaM activation and Thr 286 autophosphorylation displace the CaMKII autoregulatory domain within the catalytic lobe to activate the subunit (yellow) and to expose an α 1C tethering site. The CaMKII holoenzyme remains bound to the α 1C COOH terminus even after removal of the Ca 2+ /CaM stimulus, and CaMKII dephosphorylation produces an inactive subunit. CaMKII may remain tethered to other cytoplasmic domains of α 1C as well. High depolarization frequencies would produce a threshold level of activated/autophosphorylated CaMKII subunits that increase the P o of the channel via phosphorylation of the NH 2 and/or COOH termini (top left). At low depolarization frequencies and under the influence of phosphatase action, CaMKII activation would not be produced, favoring a low P o for α 1C (top right).

CC BY-NC-SA

no

2022-01-13 07:26:11

J Cell Biol. 2005 Nov 7; 171(3):537-547

PMC1350743

16314435

20051166f1

Figure 1. OVA-specific CD4 + CD25 + regulatory T cells regulate allergen-induced airway inflammation in vivo . OVA-sensitized mice received either 5 × 10 5 CD4 + CD25 + cells or an equivalent volume of PBS and were challenged through the airways with OVA. (A) AHR was measured 24 h after the final OVA challenge using a Buxco system in which mice were exposed to increasing concentrations of methacholine. Values are expressed as means ± SEM ( n = 9–12 mice/group from two separate experiments). *, P < 0.05 compared with OVA-sensitized mice that received PBS instead of CD4 + CD25 + cells. (B) Lungs were fixed, sectioned, and stained with hemotoxylin and eosin. Representative sections are shown for each treatment group. BAL and lung tissue digest cells (C) were isolated as described in Materials and methods, and eosinophil numbers were determined by differential counts. Values are expressed as medians ( n = 9–12 mice/group from two separate experiments). *, P < 0.05 compared with OVA-sensitized mice that received PBS instead of CD4 + CD25 + cells.

CC BY-NC-SA

no

2022-01-13 09:01:11

J Exp Med. 2005 Dec 5; 202(11):1539-1547

PMC1350743

16314435

20051166f2

Figure 2. Transfer of CD4 + CD25 + regulatory T cells reduces Th2 cell responses in the lung after allergen challenge. BAL (A) and lung tissue digest cells (B) were isolated as described in Materials and methods. Th2 cell numbers were determined 24 h after the final OVA challenge by antibody staining and flow cytometric analysis, as described in Materials and methods. Th2 cells were defined as cells that were double-stained for CD4 and the Th2 cell–specific marker T1/ST2. Data are expressed as means ± SEM. IL-5 and IL-13 levels measured in BAL fluid (C) and lung homogenate supernatant (D) by ELISA. IL-10 and active TGF-β1 levels were measured in BAL fluid (E) and lung homogenate supernatant (F) by ELISA. Data are expressed as medians ( n = 9–12 mice/group from two separate experiments). *, P < 0.05 compared with OVA-sensitized mice that received PBS instead of CD4 + CD25 + cells.

CC BY-NC-SA

no

2022-01-13 09:01:11

J Exp Med. 2005 Dec 5; 202(11):1539-1547

PMC1350743

16314435

20051166f3

Figure 3. Suppression of allergen-induced airway inflammation is IL-10 dependent. Mice were treated with anti–IL-10R antibody or control Ig during the allergen challenge phase of allergic inflammation. AHR (A) and lung eosinophilia (B) were quantified as described in Materials and methods. IL-5 (C), IL-13 (D), and IL-10 (E) levels were determined in lung homogenate supernatant by ELISA. Data are expressed as means ± SEM (A) or medians (B–E; n = 4–6 mice/group). *, P < 0.05 compared with OVA-sensitized mice that received PBS and control Ig.

CC BY-NC-SA

no

2022-01-13 09:01:11

J Exp Med. 2005 Dec 5; 202(11):1539-1547

PMC1350743

16314435

20051166f4

Figure 4. Transfer of IL-10–deficient CD4 + CD25 + regulatory T cells suppresses allergen-induced airway inflammation. Mice received either wild-type CD4 + CD25 + regulatory T cells, IL-10–deficient CD4 + CD25 + regulatory T cells, or an equivalent volume of PBS as a control. AHR (A) and lung eosinophilia (B) were determined as described in Materials and methods. IL-5 (C), IL-13 (D), and IL-10 (E) levels were measured in lung homogenate supernatant by ELISA. Data are expressed as means ± SEM (A) or medians (B-E; n = 6–11 mice/group from two separate experiments). *, P < 0.05 compared with OVA-sensitized mice that received PBS instead of CD4 + CD25 + cells.

CC BY-NC-SA

no

2022-01-13 09:01:11

J Exp Med. 2005 Dec 5; 202(11):1539-1547

PMC1350743

16314435

20051166f5

Figure 5. IL-10 is produced by CD4 + T cells during allergen-induced airway inflammation and is increased by transfer of CD4 + CD25 + regulatory T cells. Lungs were digested with collagenase and DNase as described in Materials and methods. Digest cells were stimulated by PMA/Ionomycin in the presence of Brefeldin A for 6 h. Cells were phenotyped by staining for CD4, CD8, CD11b (macrophages), CD11c (dendritic cells), and B220 (B cells). Granulocytes were defined by forward and side scatter. (A) Data are expressed as median cell types producing IL-10 with interquartile range ( n = 6–14 mice/group in three separate experiments). *, P < 0.05 compared with OVA-sensitized mice that received PBS instead of CD4 + CD25 + cells. (B) Data are shown as representative FACS plots showing costaining of lung tissue digest cells with CD4 and IL-10. Percentages in the top right quadrants refer to median percentages of CD4 cells expressing IL-10 ( n = 6–14 mice/group).

CC BY-NC-SA

no

2022-01-13 09:01:11

J Exp Med. 2005 Dec 5; 202(11):1539-1547

PMC1350947

16260490

20051376f1

Figure 1. Persistent MV infection in the transgene mice. The SSPE tg mouse model was generated by first creating tg mice expressing the MV receptor CD46 (reference 12 ) and then breeding these mice on a Rag1 −/− background (references 19 , 30 ). (A, C, and E–H) MV replication in individual 6–8-wk-old mice given MV strain Edmonston i.c. All mice died by day 65. Mean time to death ± SD of 47 ± 4 d after viral inoculation. A and C, 50×; E–H, 100×. Equivalent virus replication was seen in all such MV-infected CD46 × Rag1 −/− mice throughout the CNS. However, when mice were given 5 × 10 7 or 10 7 , but not 5 × 10 6 , syngeneic splenocytes either 3 d before or 3 d after MV challenge, 10 out of 10 mice in each group survived >200 d. (B and D) Representative image from the hippocampus (B) and cortex (D) of 10 out of 10 mice receiving 10 7 or 5 × 10 7 splenic lymphocytes 3 d before MV challenge, respectively (50×). When five Rag1 −/− or five CD46 mice alone were inoculated i.c. with 10 5 MV, all failed to express MV RNA or protein in the CNS. (I and J) Photomicrographs from an electron microscopic study of an MV-infected neuron in the cortex (I) and hippocampus (J) from a CD46 × Rag1 −/− tg mouse. The arrows in I indicate four collections of viral nucleocapsid inclusions (44,800×), whereas J shows cytoplasmic inclusions (78,000×) of loosely coiled viral nucleocapsid helices. In the CNS, only neurons were shown to contain MV antigens.

CC BY-NC-SA

no

2022-01-13 09:01:09

J Exp Med. 2005 Nov 7; 202(9):1185-1190

PMC1350947

16260490

20051376f2

Figure 2. Anatomical location and frequency of 69 A to G hypermutations. The hypermutations were recorded in the MV M gene open reading frame from one representative mouse out of five studied. 12 cDNA clones were isolated, and each position of A to G changes in the MV M is marked with an asterisk. Data showing the A to G changes from base 215 to 340 are displayed. For our initial set of five MV M sequences from this mouse, one MV M clone was isolated that possessed 20 A to G changes and also ablated a unique Alu I site within the MV M sequence. This observation allowed us to screen for additional Alu I − hypermutated M sequences from this as well as other mice. 11 additional hypermutated MV M sequences were uncovered using the Alu I screen, strongly suggesting that an MV with 20 A to G M gene changes arose, replicated, and underwent further mutation leading to a total number of 69 A to G changes. Quantitation of Alu I − versus Alu I + MV M clones from this mouse revealed that ∼40% of the MV M clones were hypermutated to A to G changes. The Alu I screen also revealed the presence and clonal expansion of hypermutated M genes in the other mice analyzed. These four other mice had 62, 91, 113, and 135 unique A to G changes, respectively. The clonal expansion of one MV M with a total of 69 A to G mutations is indicated. (bottom) Several of these mutations are shown and compared with the authentic sequence of the inoculated MV. a.a., amino acid.

CC BY-NC-SA

no

2022-01-13 09:01:09

J Exp Med. 2005 Nov 7; 202(9):1185-1190

PMC1350947

16260490

20051376f3

Figure 3. Data implicating a dual viral hit mechanism for causing persistent MV SSPE-like infection. The first hit is immunosuppressive LCMV Cl 13 given i.v. at a dose of 2 × 10 6 . (A–C) When 10 5 PFU of MV is administered i.c. 10 d later, the neurons of all inoculated mice become persistently infected with MV. LCMV Cl 13 infects DCs (references 25 , 26 ) and impairs their antigen-presenting capacity so they cannot activate naive T cells or B cells. The result is transient immunosuppression beginning at days 4–5 after LCMV inoculation; peak suppression occurs at days 10–15 and slowly diminishes thereafter, lasting for ∼60 d. A–C are from three individual mice given MV 10 d after LCMV Cl 13 inoculation and killed at 150 (A), 200 (B), and 240 (C) d, respectively, after MV inoculation. Sections are peroxidase stained with antibody to MV, and abundant brown staining neurons are indicated by arrows. (D) Presence of CD4 T cells in the leptomeninges. (E) B cells around blood vessels in the brain parenchyma. (F) CD8 T cells in the brain parenchyma by themselves. Arrowheads in D–F indicate several of these lymphocytes. CD4 and CD8 T lymphocytes and B cells were found in all three locations in four out of four mice studied 200 d after receiving MV and 210 d after receiving LCMV Cl 13.

CC BY-NC-SA

no

2022-01-13 09:01:09

J Exp Med. 2005 Nov 7; 202(9):1185-1190

PMC1351127

15824131

200411118f1

Figure 1. ZW10 and Zwint-1 reside in distinct kinetochore subcomplexes in HeLa cells. (A) Localization of ZW10 LAPtag in a HeLa cell line stably expressing the fusion protein (clone LZ5). Cells were treated with nocodazole for 30 min before fixation, and stained for ZW10 LAPtag (anti-GFP), centromeres (ACA), and DNA (DAPI). (B) Localization of Zwint-1 LAPtag in clone LINT2.8. Treatment and staining was performed as in A, except cells were preextracted before fixation. (C) Immunoblot of whole cell lysates of HeLa cells and clone LZ5. Lysates were probed for ZW10, ZW10 LAPtag (anti-GFP), and tubulin. (D and E) Tandem affinity purification of Zwint-1 LAPtag (D) and ZW10 LAPtag (E) from mitotically arrested cells. 25% of eluate was analyzed by SDS-PAGE followed by silverstain and 75% was analyzed by MudPIT mass spectrometry. Name, percent sequence coverage, and expected molecular weight of the identified proteins are indicated in the table. Suspected position of the identified proteins on the silver stained gel are indicated on the right. Unlabeled bands on silverstain likely include DC31 (∼32 kD), Q9H410 (∼40 kD), and the nonspecific proteins HSP70 (∼70 kD) and α-tubulin (∼50 kD).

CC BY-NC-SA

no

2022-01-13 07:23:19

J Cell Biol. 2005 Apr 11; 169(1):49-60

PMC1351127

15824131

200411118f2

Figure 2. Interaction between Zwint-1 and ZW10 controls ZW10 kinetochore localization. (A) Immunoblot of Zwint-1 immunoprecipitates shows weak interaction with ZW10. Cells of clone LINT2.8 were subjected to immunoprecipitation with control antibody (Con) or anti-GFP antibody to precipitate Zwint-1 LAPtag and the precipitate was analyzed for the presence of endogenous ZW10. HSS, high speed supernatant before the immunoprecipitation. Bands labeled with asterisks are background due to precipitation from HSS with the anti-GFP antibody. White line indicates that intervening lanes have been spliced out. (B) Analysis of Zwint-1 knockdown efficiency by immunoblot using cells expressing Zwint-1 LAPtag . Lysates of LINT2.8 cells untransfected or transfected with mock or Zwint-1 siRNA plasmid for 72 h were analyzed for Zwint-1 LAPtag (anti-GFP), ZW10, and tubulin expression. Percentage of remaining protein was determined by serial dilution immunoblotting. Band labeled with asterisks is protein that cross reacts with anti-GFP in the LINT2.8 cell line. (C) Immunolocalization of ZW10 in cells depleted of endogenous Zwint-1. HeLa cells transfected as in B were treated with nocodazole for 30 min before fixation and stained for endogenous Zwint-1 and ZW10, and for centromeres (ACA) and DNA (DAPI).

CC BY-NC-SA

no

2022-01-13 07:23:19

J Cell Biol. 2005 Apr 11; 169(1):49-60

PMC1351127

15824131

200411118f3

Figure 3. Characterization of Xenopus ZW10 and Rod. (A and B) Schematic alignment of Xenopus and human ZW10 (A) or Xenopus Rod COOH (XL107l09) and human Rod (B). Amino acid positions as well as percentage identity and additional (*) similarity on the protein level are indicated. (C) Coomassie staining of purified recombinant X-ZW10 (X-Z) and X-Rod COOH (X-R COOH ). (His) 6 -tagged proteins were purified from insect cells and analyzed by Coomassie blue staining. (D) Immunoblot analysis of pAbs to X-ZW10 and X-Rod COOH . 20 ng of recombinant protein (rec. prot.) and 1 μl CSF extract were analyzed by immunoblot with affinity purified anti–X-ZW10 (1348) or anti–X-Rod COOH (1351). Position of the endogenous frog proteins in the CSF extract is indicated. Cross-reacting proteins are marked by asterisks. (E) Immunolocalization of X-ZW10 and X-Rod. Sperm nuclei replicated in cycled CSF extract were immunostained for X-BubR1 and X-ZW10 or X-Rod. DNA (DAPI) is in blue. Enlarged boxes show overlap of X-BubR1 and X-ZW10–X-Rod signals on a sister kinetochore pair.

CC BY-NC-SA

no

2022-01-13 07:23:19

J Cell Biol. 2005 Apr 11; 169(1):49-60

PMC1351127

15824131

200411118f4

Figure 4. The X-ZW10–X-Rod complex is essential for establishment and maintenance of the mitotic checkpoint. (A) Immunoblot of immunodepleted CSF extract. Extracts were depleted with anti–rabbit IgG (ΔIgG), anti–X-ZW10 (ΔX-Z), or anti–X-Rod COOH (ΔX-R). 1 μl of extract or the eluate of 1-μl beads from the immunodepletion were analyzed for X-ZW10–X-Rod levels. (B) Cdk1 kinase activity in Xenopus oocyte extracts. CSF extracts, mock depleted (ΔIgG) or depleted of the X-ZW10–X-Rod complex (ΔX-ZW10), were supplemented with nocodazole and the indicated amount of sperm nuclei and mitotic checkpoint activity was measured by the ability to maintain Cdk1 kinase activity toward histone H1 (H1) after inactivation of CSF by calcium for 0, 30, or 60 min. White lines indicate that intervening lanes have been spliced out. (C) CSF extracts were supplemented with sperm (15,000 per μl of extract) before (maintenance) or after (establishment) mock depletion (ΔIgG) or depletion of the X-ZW10–X-Rod complex (ΔX-ZW10 or ΔX-Rod). Mitotic checkpoint activity was measured as in B.

CC BY-NC-SA

no

2022-01-13 07:23:19

J Cell Biol. 2005 Apr 11; 169(1):49-60

PMC1351127

15824131

200411118f5

Figure 5. X-ZW10–X-Rod regulate kinetochore localization of X-BubR1, X-Mad1, and X-Mad2. (A–C) Immunolocalization of checkpoint proteins in depleted Xenopus extracts. Unreplicated sperm nuclei in mock (A, ΔIgG) or X-ZW10–X-Rod–depleted (B, ΔX-ZW10; or C, ΔX-Rod) extracts were immunostained with antibodies to the indicated proteins and X-BubR1. DNA (DAPI) is in blue. (D) Immunoblot of various checkpoint proteins in Xenopus extracts depleted of the X-ZW10–X-Rod complex. CSF extracts or checkpoint activated extracts were mock depleted (ΔIgG) or depleted of the X-ZW10–X-Rod complex (ΔX-Z or ΔX-R) and analyzed for presence of the indicated checkpoint proteins. (E) Immunolocalization of X-ZW10 and X-Rod in X-BubR1–depleted extracts. CSF extracts, mock depleted (ΔIgG) or depleted of X-BubR1 (ΔX-BubR1) were analyzed for kinetochore localization of X-ZW10 and X-Rod. DNA (DAPI) is in blue.

CC BY-NC-SA

no

2022-01-13 07:23:19

J Cell Biol. 2005 Apr 11; 169(1):49-60

PMC1351127

15824131

200411118f6

Figure 6. Human ZW10 is essential for mitotic checkpoint signaling. (A) Immunoblot analysis of ZW10 knockdown. HeLa cells were transfected with control or ZW10 siRNA duplexes. 5 d after transfection, total lysates were analyzed for ZW10 and tubulin protein. Percentage knockdown was determined by serial dilution immunoblotting. (B) Immunofluorescence analysis of ZW10 knockdown. Cells were transfected as in A, treated with nocodazole for 30 min before fixation and stained for ZW10 and DNA (DAPI). (C) Flow cytometric analysis of the fraction of phospho-histone H3-positive cells of mock siRNA, ZW10 siRNA and Zwint-1 siRNA cells 96 h after introduction of the siRNAs. Cells were transfected as in A, left untreated or treated with nocodazole for 16 h, and the entire population was analyzed for phospho-histone-H3 (y axis) and DNA (propidium iodide, x axis). Dot plots represent 4×10 4 cells for control siRNA and 10 4 cells for ZW10 or Zwint-1 siRNA. Percentages indicate fraction of cell population that is phospho-histone H3 positive. (D) Average fold increase of phospho-histone H3 staining after nocodazole treatment. Cells were transfected, treated, and analyzed as in C. Graph represents average of four independent experiments. (E) Graph of colony outgrowth assay. Cells were transfected as in A and retransfected for 7 d after which the colonies were stained and counted. Graph represents average of three experiments. (F and G) Aberrant mitosis in cells lacking ZW10. Cells were transfected as in A and stained with DAPI. Shown are typical interphase nuclei. (F) Cells transfected with ZW10 siRNA. (G) Cells transfected with control siRNA.

CC BY-NC-SA

no

2022-01-13 07:23:19

J Cell Biol. 2005 Apr 11; 169(1):49-60

PMC1351127

15824131

200411118f7

Figure 7. Mad1 and Mad2 are unable to bind kinetochores in absence of ZW10. (A–F) Immunolocalization of various proteins in ZW10-depleted cells. HeLa cells were transfected as in Fig. 6 A and treated with nocodazole 30 min before fixation. Cells were stained with ACA (A, B, and C), ZW10 (A, B, D, and F), and dynein intermediate chain (IC) (A), Mad1 (B), Mad2 (C), Bub1 (D), or BubR1 (F). DNA (DAPI) is in blue. Enlarged boxes show a pair of kinetochores. (E) Quantification of kinetochore fluorescence. Normalized integrated intensity (see Materials and methods) of Mad1, Mad2, and Bub1 is shown in mock siRNA and ZW10 siRNA cells. Error bars indicate the SD from measurements of three cells. (G) Model for function of the ZW10–Rod complex in mitotic checkpoint signaling. The ZW10-interactor Zwint-1 is in a structural kinetochore complex with Ndc80–HEC1 and Mis12 that is linked to the inner kinetochore by KNL-1 AF15q14 . The ZW10–Rod–Zwilch complex associates dynamically with the unattached kinetochore through interaction with Zwint-1, where it regulates kinetochore binding of the Mad1–Mad2 heterodimer and thus activation of Mad2.

CC BY-NC-SA

no

2022-01-13 07:23:19

J Cell Biol. 2005 Apr 11; 169(1):49-60

PMC1351290

15753208

20041470f1

Figure 1. Serum reconstitution of J H mice. Lesion size of J H mice (closed triangles) and BALB/c mice (closed circles) was compared with J H mice (open triangles) and BALB/c mice (open circles) given 200 μl of antisera to L. major on days 1, 7, and 14. Parasite burdens (inset) in BALB/c and J H mice that were infected with (diagonally striped bars) or without (black bars) αLm (α– L. major ) antisera were determined on day 35 by limiting dilution assays as described previously (reference 3 ). Error bars represent the standard error of the mean of three separate experiments done with a minimum of four mice per group.

CC BY-NC-SA

no

2022-01-13 09:00:57

J Exp Med. 2005 Mar 7; 201(5):747-754

PMC1351290

15753208

20041470f2

Figure 2. Serum reconstitution of J H mice 3 wk after infection. Lesion size of J H mice (closed circles) were compared with those of J H mice given 200 μl αLm (α– L. major ) antiserum on day 21 after infection (open circles). Parasite burdens (inset) were determined at 42 d after infection by limiting dilution assays as described previously (reference 3 ). Error bars represent the standard deviation of the mean of four determinations. This experiment is representative of three.

CC BY-NC-SA

no

2022-01-13 09:00:57

J Exp Med. 2005 Mar 7; 201(5):747-754

PMC1351290

15753208

20041470f3

Figure 3. Cytokine production in mice administered anti– L. major antiserum. (A) Cytokine production by lymph node T cells from infected J H mice (white bars) was compared with J H mice administered 200 μl αLm (α– L. major ) antiserum on days 1, 7, and 14 after infection (black bars). Lymph nodes were removed on day 21 and stimulated with soluble leishmania antigen. Supernatants were harvested 72 h later and assayed for IFN-γ and IL-4 by ELISA. (B) IL-10 production in lesions of J H mice. IL-10 protein (left) and mRNA (right) were determined in two groups of J H mice infected with 2 × 10 5 L. major amastigotes. On day 21 of infection, one group was administered 600 μg of purified αLm-IgG i.p. IL-10 protein levels in the lesions of three infected mice were measured by ELISA 4 d after the administration of αLm-IgG (left axis). RNA was isolated from footpad lesions on day 2 after IgG administration, and real-time PCR was performed to determine relative IL-10 mRNA (right axis). Levels represent the average from three infected mice, and mRNA was normalized to hypoxanthine phosphoribosyltransferase levels in a single infected foot.

CC BY-NC-SA

no

2022-01-13 09:00:57

J Exp Med. 2005 Mar 7; 201(5):747-754

PMC1351290

15753208

20041470f4

Figure 4. IgG reconstitution of J H mice and the effect of α–IL-10R mAb. Three parallel groups of J H mice were infected with 2 × 10 5 L. major amastigotes. One group (open circles) was administered 600 μg of purified αLm-IgG i.p. on days 1, 8, and 15. Another group (gray triangles) was given the same dose of αLm-IgG, along with α–IL-10R on days 0 (1 mg), 7 (200 μg), and 14 (200 μg). The third group (closed circles) received no treatment. Lesion size was measured at semi-weekly intervals. Parasite burdens (inset) were determined by limiting dilution. Error bars represent the standard deviation of the mean.

CC BY-NC-SA

no

2022-01-13 09:00:57

J Exp Med. 2005 Mar 7; 201(5):747-754

PMC1351290

15753208

20041470f5

Figure 5. The immunization of C57BL/6 mice with OVA. Lesion development in OVA-immunized mice (open circles) was compared with control mice given IFA alone (closed circles). Immunized mice were given 25 μg OVA in 500 μL of IFA and boosted 2 wk later. Both groups of mice were infected with L. major resuspended in PBS containing 50 μg/ml OVA. Parasite burdens (inset) were determined on day 34. Error bars represent the standard deviation of the mean.

CC BY-NC-SA

no

2022-01-13 09:00:57

J Exp Med. 2005 Mar 7; 201(5):747-754