Prefoldin is a hexameric chaperone that facilitates posttranslational folding of actins and other cytoskeletal proteins by the Tcp1-containing ring complex chaperonin, TriC. The present study characterized mice with a null mutation in Pfdn1, which encodes the first subunit of the Prefoldin complex. Pfdn1-deficient mice displayed phenotypes characteristic of defects in cytoskeletal function, including manifestations of ciliary dyskinesia, neuronal loss, and defects in B and T cell development and function. B and T cell maturation was markedly impaired at relatively early stages, namely at the transitions from pre-pro-B to pre-B cells in the bone marrow and from CD4CD8 double-negative to CD4+CD8+ double-positive T cells in the thymus. In addition, mature B and T lymphocytes displayed cell activation defects upon Ag receptor cross-linking accompanied by impaired Ag receptor capping in B cells. These phenotypes illustrate the importance of cytoskeletal function in immune cell development and activation.

Actins and tubulins, as ubiquitous components of the cytoskeleton, play essential roles in fundamental cellular processes including motility, macromolecular transport, signal transduction, and cell division (1). Actin and tubulin subunits assemble in a head-to-tail manner by a regulated, energy-consuming process (2, 3). Fiber asymmetry enables actin and tubulin cytoskeletons to orchestrate rapid and spatially localized changes in cell structure and function involved in diverse processes from axon outgrowth to immune synapse signaling (4, 5).

Efficient synthesis of actin and tubulin subunits requires two protein complexes, Prefoldin and TRiC/CCT (Tcp1 ring complex/chaperonin-containing Tcp1) that facilitate protein folding (reviewed in Ref. 6). Prefoldin is a molecular chaperone consisting of six radially organized proteins, each forming a coiled-coil tentacle that participates in substrate binding (7, 8). Prefoldin was first identified from genetic interactions implicating the complex in cytoskeleton biosynthesis (9) and as a biochemical activity that transferred denatured β-actin to TRiC (10). TRiC, a cytoplasmic chaperonin, transiently interacts with ∼10% of cellular proteins and folds several essential proteins, including actins and tubulins, by an ATP-dependent mechanism (reviewed in Ref. 11). Nascent β-actin binds Prefoldin during translation (12) and is presented to the TRiC chaperonin complex (8, 10). Although TRiC can bind protein substrates directly, Prefoldin significantly enhances the rate of TRiC-mediated protein folding (13).

Unlike TRiC, yeast Prefoldin (GimC) is not essential for cell viability (9, 10). However, GimC mutations generate cytoskeletal defects similar to temperature-conditional TRiC mutations (9, 10), providing further evidence for biochemical cooperation between the chaperone and chaperonin. Prefoldin subunits 2, 3, and 6 influence Caenorhabditis elegans development (14, 15); thus, despite evidence of limited promiscuity among chaperones with regard to substrate binding and chaperonin presentation (16), nonredundant activities of Prefoldin appear to be required for cytoskeletal function in lower eukaryotes.

Genetic analysis of mammalian Prefoldin is expected to generate phenotypes indicative of biologically important folding substrates in mammalian cells. In particular, we would like to know whether Prefoldin mutant phenotypes are consistent with aberrant cytoskeletal function, as in yeast, or possess defects involving a wider range of protein substrates. Mice deficient in Prefoldin chaperone functions may also clarify the involvement of the cytoskeleton (17, 18) or other misfolded proteins in aging-associated diseases (19, 20, 21, 22). Finally, the availability of Prefoldin-deficient cells and animals is expected to facilitate functional studies of the chaperone in different biological processes.

In the present study, we characterized a null mutation in the gene (Pfdn1) for Prefoldin subunit 1 induced by gene trapping in mouse embryonic stem (ES)5 cells. We show that Pfdn1-deficient mice develop to term but display severe abnormalities characteristic of defects in cytoskeletal function, including ciliary dyskinesia, loss of neuron tracts in the brain, and defects in B and T cell development and function similar to but more severe than Wiskott-Aldrich syndrome protein (WASP) and WASP-interacting protein (WIP) mutations. These phenotypes appear to reflect processes such as immune cell signaling and axon guidance that place particular demands on cytoskeletal synthesis and remodeling.

The construction of a GTR1.3 retrovirus gene trap library, as well as the analysis of 3′ RACE products, has been described (23).

The insertion site was located by inverse PCR. Briefly, 50–200 ng of genomic DNA from mouse tail was digested by AvaII (New England Biolabs) and 20–50 ng from the digest was ligated in a 20-μl reaction (T4 DNA ligase and 10× ligase buffer with 1 mM ATP (New England Biolabs)) at room temperature for 10 min. Two microliters of the ligation product (p) was amplified by PCR in 50-μl reaction (Roche) with the upstream (U) and downstream (D) primers 5′-CAGTCCTCCGATTGACTGAG-3′ (Dp) and 5′-GGGGTTGTGGGCTCTTTAT-3′ (Up). The amplification protocol consisted of 35 cycles incubating at 94°C for 1 min, 55°C for 1 min, and 72 °C for 1 min.

Mice and cells were genotyped by PCR analysis. Twenty to 50 ng of genomic DNA (g) was used as template in a 50-μl reaction using the Roche PCR mix with the upstream (U) and downstream (D) primers 5′-TGGGATAATGCCCACAGGTA-3′ (Dg), 5′-AAGCACTCAGAGCAGCAAGTT-3′ (Ug), and 5′-CAGTCCTCCGATTGACTGAG-3′ (Dp). Samples were amplified through 30 cycles of incubation at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min.

The expression of Pfdn1 mRNA transcripts in mice and cells was assessed by Northern blot hybridization to a [32P]dCTP-labeled probe derived from the full length Pfdn1 cDNA, as described previously (24).

Prefoldin 1 protein expression in ES cell clones was assessed by Western blot analysis. Cell lysates were fractionated by electrophoresis on a 12% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (PerkinElmer Life Science). Prefoldin 1 proteins were detected by using donkey polyclonal Abs raised against an internal region of the protein (Santa Cruz Biotechnology), as described previously (24).

Flow cytometric analyses were performed for the phenotypic characterization of B and T lymphocytes as described (25, 26). Single-cell suspensions were prepared from lymphoid organs, and T cell populations in the thymus and spleens were identified by anti-CD4, anti-CD8, and anti-TCRβ. B cell subpopulations in the bone marrow (BM) and spleen were identified by staining with anti-BP-1 (Ly-51), anti-CD25, anti-HSA, biotinylated anti-CD43 (S7), anti-B220, anti-CD23, anti-CD21, anti-CD19, anti-IgM, anti-IgD, anti-CD5 (Ly-1), and streptavidin-allophycocyanin (purchased from BD Pharmingen) and anti-AA4.1 (purchased from eBiosciences). Data were collected on an BD LSR II cytometer (BD Biosciences) and analyzed using FlowJo (Tree Star).

For in vitro activation assays, splenocytes were isolated by hypotonic lysis of RBC and cultured at 1 × 106 cells/ml in RPMI 1640 medium supplemented with 10% FBS (HyClone), 2 mM glutamine, 50 μM 2-ME, and 100 U/ml penicillin/streptomycin with or without F(ab′)2 goat anti-mouse IgM (Jackson ImmunoResearch) and anti-CD3 for 18 h. Cells were also incubated with PMA and ionomycin (100 nM each) as positive controls. B cell activation was determined by cell surface staining with anti-CD19 (allophycocyanin) and anti-CD86 (FITC). T cell activation was determined by cell surface staining with anti-CD4 (FITC) and anti-CD69 (PE). Stained cells were incubated with 7-aminoactinomycin D (Molecular Probes) immediately before acquisition, enabling the resolution of live vs dead cells by flow cytometry. Data were collected on an BD LSR II cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Embryos at embryonic day 13.5 were isolated, minced, and treated with 2.5% trypsin-EDTA (Invitrogen). Cell suspensions were washed and cultured in DMEM (Mediatech), supplemented with 10% FCS (heat inactivated at 55°C for 30 min) and 100 U/ml penicillin-streptomycin (Invitrogen). Primary mouse embryonic fibroblasts (MEFs) were cultured to near-confluence densities and passed at 1:2 ratios until cell lines were obtained.

Heterozygous control and Pfdn1−/− splenocytes (1 × 106) were stimulated with 10 μg/ml Cy5-conjugated goat anti-mouse (F(ab′)2) IgM (Jackson ImmunoResearch) in 100 μl PBS with 2% FCS for the indicated times at 37°C. Cells were cytospun onto poly-l-lysine-coated glass slides at 500 rpm for 3 min, fixed in acetone and methanol, and washed with PBS. Cells were visualized using a Zeiss LSM510 inverted laser scanning microscope (×40 original magnification, 1.3 numerical aperture objective). Images were acquired using argon and HeNe2 lasers, and the extent of capping in IgM+ B cells (50 to 200) was determined visually and scored in a blind manner by an experienced microscopist. A B cell was scored capped if the BCR polarized on half or less than half of the circumference of the cell. Only IgM+ cells were counted as B cells in the splenocytes used in imaging experiments.

A mutation in the Pfdn1 gene (Fig. 1,A) was identified in a screen of ES cell clones mutagenized by the GTR1.3 gene trap vector (23). The disrupted gene was identified by sequencing Neo-fusion transcripts amplified by 3′ RACE, which, in the case of the Pfdn1 mutation, spliced to the fourth and last exon of the gene (Fig. 1,B), thus deleting the coiled-coil domain required for interactions between Prefoldin and peptide substrates (7). The exact location of the provirus in the third intron was subsequently determined by sequencing the region of genomic DNA adjacent to the targeting vector cloned by inverse PCR (Fig. 1 C).

FIGURE 1.

Pfdn1 gene disruption. A, The GTR1.3 gene trap retrovirus was inserted into the third intron of the Pfdn1 gene as shown. The insert enables upstream Pfdn1 sequences to splice to a 3′ exon consisting of the 3′ end of a puromycin resistance gene (3′ Puro), a β-galactosidase reporter (lacZ), and a polyadenylation signal (PA), whereas Neo sequences are expressed from the promoter of the RNA polymerase 2 gene (Pol2) to splice to exon 4. Primers complementary to proviral (Dp and Up) and cellular (Ug and Dg) sequences used for genotyping are indicated (where D is downstream, U is upstream, p is provirus DNA, and g is genomic DNA). B, Sequence of GTR1.3-Pfdn1 fusion transcripts. Cellular sequences appended to Neo fusion transcripts were cloned by 3′ RACE and sequenced, revealing an insert in the Pfdn1 gene. C, Genomic sequences upstream of the GTR1.3 provirus were amplified by inverse PCR and sequenced, providing the exact location of the provirus in the Pfdn1 gene. D, Genotype analysis. Wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) mice were genotyped by PCR using a mixture of Ug, Dg, and Dp primers. M, Marker. E, Western blot analysis of Pfdn1 expression. Embryonic fibroblasts from mice with the indicated genotypes were analyzed by Western blot analysis using Abs against Pfdn1 (left panel) or heterogenous nuclear ribonuclear protein (hnRNP) C (right panel). F, Northern blot analysis of Pfdn1 expression. Ten micrograms of RNA from mouse embryonic fibroblasts with the indicated genotypes were probed with Pfdn1-specific (left panel) or Gapdh-specific (right panel) sequences.

FIGURE 1.

Pfdn1 gene disruption. A, The GTR1.3 gene trap retrovirus was inserted into the third intron of the Pfdn1 gene as shown. The insert enables upstream Pfdn1 sequences to splice to a 3′ exon consisting of the 3′ end of a puromycin resistance gene (3′ Puro), a β-galactosidase reporter (lacZ), and a polyadenylation signal (PA), whereas Neo sequences are expressed from the promoter of the RNA polymerase 2 gene (Pol2) to splice to exon 4. Primers complementary to proviral (Dp and Up) and cellular (Ug and Dg) sequences used for genotyping are indicated (where D is downstream, U is upstream, p is provirus DNA, and g is genomic DNA). B, Sequence of GTR1.3-Pfdn1 fusion transcripts. Cellular sequences appended to Neo fusion transcripts were cloned by 3′ RACE and sequenced, revealing an insert in the Pfdn1 gene. C, Genomic sequences upstream of the GTR1.3 provirus were amplified by inverse PCR and sequenced, providing the exact location of the provirus in the Pfdn1 gene. D, Genotype analysis. Wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) mice were genotyped by PCR using a mixture of Ug, Dg, and Dp primers. M, Marker. E, Western blot analysis of Pfdn1 expression. Embryonic fibroblasts from mice with the indicated genotypes were analyzed by Western blot analysis using Abs against Pfdn1 (left panel) or heterogenous nuclear ribonuclear protein (hnRNP) C (right panel). F, Northern blot analysis of Pfdn1 expression. Ten micrograms of RNA from mouse embryonic fibroblasts with the indicated genotypes were probed with Pfdn1-specific (left panel) or Gapdh-specific (right panel) sequences.

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ES cells with the Pfdn1 mutation produced germline chimeras after injection into C57BL/6 blastocysts. Mice containing the mutated allele were genotyped by PCR using a mixture of primers complementary to the flanking genomic DNA sequences and to the LTR (Fig. 1,D). Pfdn1 expression was completely disrupted in homozygous mutant MEFs as assessed by Western blot analysis. Thus, the 20-kDa Pfdn1 protein detected in wild-type and heterozygous MEFs was not detected in homozygous mutant MEFs (Fig. 1,E). In addition, homozygous mutant MEFs did not express wild-type Pfdn1 transcripts but did express lower levels of a larger RNA fragment, presumably generated by splicing of the upstream exons to the 3′ Puro-LacZ sequence carried by the provirus (Fig. 1 F). Although these larger transcripts have the potential to express aberrant Pfdn1 proteins, none was detected by Western blot analysis. Thus, the gene entrapment appeared to produce a null mutation.

Pfdn1−/− mice, although produced at a Mendelian ratio, were smaller in size than wild-type and heterozygous littermates (Fig. 2,A), grew more slowly (Fig. 2,B), and most died before 5 wk of age (Fig. 2 C). Some homozygous mutants were visibly smaller at birth. The average weights of mutant mice at 2, 3, and 4 wk of age were 3.9 ± 0.6 g (n = 14), 5.3 ± 1.0 g (n = 13), and 4.9 ± 1.0 g (n = 10), respectively, while the wild-type and heterozygous littermates weighed 8.3 ± 0.8 g (n = 26), 12.7 ± 1.8 g (n = 22), and 15.7 ± 1.7 g (n = 17), respectively. Pfdn1−/− mice lost an average of 0.6 ± 0.2 g (n = 5) during the week before death, indicating physical wasting. In addition, ∼40% of the homozygous mutant mice were observed with uncoordinated movement, indicating neuromuscular dysfunction.

FIGURE 2.

Growth and survival of Pfdn1 mutant mice. A, Appearance of wild-type (left) and mutant (right) mice at 3 wk of age. B, Average weights of wild-type and heterozygous mice (gray) and homozygous mutant mice (black) at 2 to 4 wk of age. C, Fraction of wild-type and heterozygous mice (gray) and homozygous mutant mice (black) surviving through 5 wk of age.

FIGURE 2.

Growth and survival of Pfdn1 mutant mice. A, Appearance of wild-type (left) and mutant (right) mice at 3 wk of age. B, Average weights of wild-type and heterozygous mice (gray) and homozygous mutant mice (black) at 2 to 4 wk of age. C, Fraction of wild-type and heterozygous mice (gray) and homozygous mutant mice (black) surviving through 5 wk of age.

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All of the homozygous mutant mice examined from 3 days to 5 wk in age were runted and displayed abnormalities in the brain and spleen. However, skeletal muscles, eyes, teeth, livers, kidneys, gut, hearts, and lungs of the mutants appeared normal. Brain abnormalities included hydrocephaly and neuronal loss affecting major commissures of the cerebrum and cerebellum. Although these changes, together with the hydrocephaly, made the cerebrums of mutant animals fragile and difficult to section, the corpus callosum in 2- and 4-wk old mutant mice were disorganized and hypocellular (Fig. 3, A and B). The arbor vitae (Fig. 3, C and D) and cerebellar commissures (data not shown) were also severely disorganized and hypocellular.

FIGURE 3.

Histology of Pfdn1-deficient mice. A–D, CNS defects. H&E-stained coronal sections through the cerebrum of wild-type (A) and homozygous mutant (B) mice and sagittal sections through the cerebellum of wild-type (C) and mutant (D) mice. The corpus callosum (CC) and arbor vitae (av) are indicated. E and F, Sinus infections. The sinuses of mutant (F) but not wild-type (E) mice were plugged with mucus and often showed signs of massive infection. G and H, Spleen abnormalities. Sections of spleens (8 μM) from wild-type (G) and mutant (H) mice stained with H&E. Spleens from mutant mice were smaller and displayed disorganized red pulp and follicles.

FIGURE 3.

Histology of Pfdn1-deficient mice. A–D, CNS defects. H&E-stained coronal sections through the cerebrum of wild-type (A) and homozygous mutant (B) mice and sagittal sections through the cerebellum of wild-type (C) and mutant (D) mice. The corpus callosum (CC) and arbor vitae (av) are indicated. E and F, Sinus infections. The sinuses of mutant (F) but not wild-type (E) mice were plugged with mucus and often showed signs of massive infection. G and H, Spleen abnormalities. Sections of spleens (8 μM) from wild-type (G) and mutant (H) mice stained with H&E. Spleens from mutant mice were smaller and displayed disorganized red pulp and follicles.

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Blood cell counts were altered in Pfdn1−/− mice, which had fewer numbers of both red (0.68 ± 0.1 vs 1.0 ± 0.03 × 109 cells/ml) and white cells (0.25 ± 0.15 vs 0.65 ± 0.1 × 106 cells/ml), but hemoglobin levels appeared normal. Micronuclei were prominent in ∼2% of the mutant erythrocytes as compared with <0.1% of the RBC in wild-type and heterozygous littermates (data not shown). Neutrophils from mutant mice also contained more extensively segmented nuclei (data not shown). Finally, the sinuses of mutant mice were filled with mucus and ∼40% of the mice showed signs of massive infection with cellular infiltrates and damage to the surrounding epithelium (Fig. 3, E and F).

Spleens from Pfdn1-deficient mice were significantly smaller than normal, weighing 83 ± 4.4% less than the spleens from littermate controls. Moreover, the histology of mutant spleens was abnormal in that lymphoid follicles were not clearly defined and the surrounding red pulp was disorganized (Fig. 3, G and H). Total cell numbers were reduced to 12 ± 7.1% of normal in 16–25 day old mice (data not shown), and the number of B cells was similarly reduced (Table I). The differences in spleen size and cellularity were significant even after factoring in the sizes of mutant animals, which were 36 ± 12% smaller than normal.

Table I.

Splenic B cellsa

B cellsT1T2Mature FoBMZ
Percentage (%)No.Percentage (%)No.Percentage (%)No.Percentage (%)No.Percentage (%)No.
Control (n = 8) 60.5 ± 8.0 16.6 ± 7.4 15.1 ± 2.3 2.5 ± 1.2 22.1 ± 5.0 3.7 ± 1.9 31.1 ± 10.7 5.1 ± 2.6 4.5 ± 0.8 2.5 ± 1.2 
Pfdn1 null (n = 9) 32.5 ± 12.2 1.1 ± 1.5 5.8 ± 5.0 0.065 ± 0.108 19.9 ± 4.3 0.271 ± 0.409 42.1 ± 10.7 0.454 ± 0.673 15.0 ± 3.9 0.131 ± 0.152 
B cellsT1T2Mature FoBMZ
Percentage (%)No.Percentage (%)No.Percentage (%)No.Percentage (%)No.Percentage (%)No.
Control (n = 8) 60.5 ± 8.0 16.6 ± 7.4 15.1 ± 2.3 2.5 ± 1.2 22.1 ± 5.0 3.7 ± 1.9 31.1 ± 10.7 5.1 ± 2.6 4.5 ± 0.8 2.5 ± 1.2 
Pfdn1 null (n = 9) 32.5 ± 12.2 1.1 ± 1.5 5.8 ± 5.0 0.065 ± 0.108 19.9 ± 4.3 0.271 ± 0.409 42.1 ± 10.7 0.454 ± 0.673 15.0 ± 3.9 0.131 ± 0.152 
a

Number of cells given in millions. FoB, Follicular B cell.

The proportion of splenic B cells was reduced (∼2–3-fold) in mutant mice with a corresponding increase in the proportion of T cells (Fig. 4,A, upper panels). Specifically, only 14–33% of the splenocytes from nine mutant mice examined were B220 positive as compared with 50–80% for wild-type or heterozygous littermates (Fig. 4,A, upper panels, and Table I), and CD4+ T cells were favored at the expense of CD8+ T cells (Fig. 4,A, lower panels; CD4:CD8 ratio 8:1; Pfdn1−/−:wild type, 2:1). Analysis of B220+ splenic B cells revealed disproportionate reductions in immature B cells, especially of the T1 type (IgMhighIgDlowAA4.1high; Fig. 4,B and Table I), modest increases in follicular B cells, and large increases in marginal zone (MZ) B cells (Fig. 4, B and C, and Table I). Although MZ B cells were significantly increased in the spleen, the peritoneal cavity was virtually devoid of both B1 and B2 B cells (Fig. 4 D). Together, these results suggests that the generation of T and B cell precursors, which occurs primarily in the BM, and/or their migration to the spleen was affected to a greater extent than their maturation in the spleen.

FIGURE 4.

FACS analysis of splenic and peritoneal lymphocytes. A, B and T cell subpopulations. Spleens from mutant (Pfdn1−/−) mice contained proportionally more T cells (top two left panels) favoring CD4+ cells (bottom two left panels) than spleens from wild-type (wt) mice. B and C, B cell subpopulations. Prefoldin 1 deficiency preferentially reduced the proportion of immature transitional 1 (T1; AA4.1high) B cells, whereas the proportion of mature follicular B cells (FoB; CD19+IgMlow/IgDhigh) (top panels in B) and MZ B cells (CD19+IgMhighCD21high/CD23; bottom panels in B and C) were significantly increased. D, FACS analysis of peritoneal B cell subpopulations. The peritoneal cavities of Pfdn1−/− mice contained fewer conventional B2 as well as B1 B cells. The data shown are representative of more than three separate experiments.

FIGURE 4.

FACS analysis of splenic and peritoneal lymphocytes. A, B and T cell subpopulations. Spleens from mutant (Pfdn1−/−) mice contained proportionally more T cells (top two left panels) favoring CD4+ cells (bottom two left panels) than spleens from wild-type (wt) mice. B and C, B cell subpopulations. Prefoldin 1 deficiency preferentially reduced the proportion of immature transitional 1 (T1; AA4.1high) B cells, whereas the proportion of mature follicular B cells (FoB; CD19+IgMlow/IgDhigh) (top panels in B) and MZ B cells (CD19+IgMhighCD21high/CD23; bottom panels in B and C) were significantly increased. D, FACS analysis of peritoneal B cell subpopulations. The peritoneal cavities of Pfdn1−/− mice contained fewer conventional B2 as well as B1 B cells. The data shown are representative of more than three separate experiments.

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Thymuses from mutant mice were smaller by weight and cell number (9.3 ± 2.7% and 4.3 ± 2.3% of normal, respectively) but were histologically normal in appearance (data not shown). As before, the size reductions were greater than the overall reduction in body weight (31.8 ± 6.4% of normal). The decreased cellularity disproportionately involved CD4+CD8+ double-positive (immature) thymocytes (10% in Pfdn1−/− vs 80% in wild type; Fig. 5,A and Table II). The accumulation of CD4CD8 double-negative cells (5% in Pfdn1−/− vs 2–3% in wild type; Table II) in most Pfdn1-deficient mice suggests a maturation defect in the transition from double-negative to double-positive T cells. By contrast, an increase in thymic CD4+ and CD8+ mature T cells and the presence of mature TCRβ+ T cells in the spleen suggests that T cell development beyond the CD4+CD8+ stage is largely unaffected.

FIGURE 5.

FACS analysis of thymic and BM lymphocytes. A, Prefoldin 1 deficiency reduces the proportion of double-positive (CD4+CD8+) immature T cells and increases the double-negative (DN) immature T cells in the thymus. The developmental block appears to occur beyond the DN1 stage as analyzed by CD44/CD25 profile of cells within the DN lymphocyte gate (bottom panels). wt, Wild type. B, FACS analysis of BM lymphocytes. IgM/B220 and CD25/B220 profiles (top and middle panels, respectively) demonstrate relative losses of B cell precursors and increases in the proportion of pro-B cells (CD25low/B220) at the expense of pre-B cells (CD25high/B220) (bottom panels). The developmental block appeared to occur at the earliest stage of B cell development as assessed by CD24/BP-1 profiles. C, The BM of mutant mice displayed higher proportions of fraction A (Fra A) cells (CD24low/BP-1low) and lower proportions of fraction C and C′ cells (CD24low/BP-1high). The data shown are representative of more than three separate experiments.

FIGURE 5.

FACS analysis of thymic and BM lymphocytes. A, Prefoldin 1 deficiency reduces the proportion of double-positive (CD4+CD8+) immature T cells and increases the double-negative (DN) immature T cells in the thymus. The developmental block appears to occur beyond the DN1 stage as analyzed by CD44/CD25 profile of cells within the DN lymphocyte gate (bottom panels). wt, Wild type. B, FACS analysis of BM lymphocytes. IgM/B220 and CD25/B220 profiles (top and middle panels, respectively) demonstrate relative losses of B cell precursors and increases in the proportion of pro-B cells (CD25low/B220) at the expense of pre-B cells (CD25high/B220) (bottom panels). The developmental block appeared to occur at the earliest stage of B cell development as assessed by CD24/BP-1 profiles. C, The BM of mutant mice displayed higher proportions of fraction A (Fra A) cells (CD24low/BP-1low) and lower proportions of fraction C and C′ cells (CD24low/BP-1high). The data shown are representative of more than three separate experiments.

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Table II.

Thymic T cells

CD4CD8 (%)CD4+CD8+ (%)CD4+ (%)CD8+ (%)
Control (n = 5) 2.7 ± 0.7 77.7 ± 5.5 14.6 ± 4.0 4.9 ± 1.1 
Pfdn1 null (n = 5) 5.2 ± 3.8 10.9 ± 3.1 65.5 ± 2.9 18.2 ± 4.5 
CD4CD8 (%)CD4+CD8+ (%)CD4+ (%)CD8+ (%)
Control (n = 5) 2.7 ± 0.7 77.7 ± 5.5 14.6 ± 4.0 4.9 ± 1.1 
Pfdn1 null (n = 5) 5.2 ± 3.8 10.9 ± 3.1 65.5 ± 2.9 18.2 ± 4.5 

Consistent with a reduction in the B cell populations in the spleen and peritoneal cavity, the total number of B220+ cells in the mutant BM (2.8 × 104 cells per gram of body weight) was significantly lower than in control animals (0.99 × 106 cells per gram), and the greatest reductions were seen in the proportion of B220+CD25+ pre-B cells, which ranged from 20 to 30% in mutant mice compared with 60–80% in control littermates (Fig. 5,B and Table III). Closer examination of the pro-B population in the BM by the Hardy fractionation scheme (27) revealed a developmental block at the earliest stage of B cell development, producing a large increase in the proportion of fraction A or pre-pro-B cells (Fig. 5 C). Thus, the production of B cell precursors in the BM is severely impaired in Pfdn1-deficient mice.

Table III.

Bone marrow B cells

B220+ (%)B220+ IgM-Pre-Pro-B (%)CD25 Pro-B (%)aCD25+ Pre-B (%)aB220+IgM+ Immature B (%)B220+IgMhigh Transitional B (%)B220highIgM+ Mature Recirculating B (%)
Control (n = 7) 56.6 ± 6.7 35.1 ± 5.1 28.2 ± 5.2 69.1 ± 4.1 6.4 ± 1.0 7.4 ± 1.6 2.5 ± 1.2 
Pfdn1 null (n = 8) 11.7 ± 6.1 7.2 ± 5.2 65.3 ± 11.8 21.3 ± 13.3 0.786 ± 0.537 1.1 ± 0.6 0.789 ± 0.629 
B220+ (%)B220+ IgM-Pre-Pro-B (%)CD25 Pro-B (%)aCD25+ Pre-B (%)aB220+IgM+ Immature B (%)B220+IgMhigh Transitional B (%)B220highIgM+ Mature Recirculating B (%)
Control (n = 7) 56.6 ± 6.7 35.1 ± 5.1 28.2 ± 5.2 69.1 ± 4.1 6.4 ± 1.0 7.4 ± 1.6 2.5 ± 1.2 
Pfdn1 null (n = 8) 11.7 ± 6.1 7.2 ± 5.2 65.3 ± 11.8 21.3 ± 13.3 0.786 ± 0.537 1.1 ± 0.6 0.789 ± 0.629 
a

Calculated from the B220+IgM pre-pro-B gate.

The effect of Pfdn1 status on signal-induced cytoskeletal reorganization was analyzed in B cells by a capping assay. Spleen cells from null mutant and heterozygous control mice were stained with fluorescent anti-mouse IgM F(ab′)2 Abs. Control B cells displayed the expected progression from cell circumference staining or local patches to a polarized BCR or cap within 5 min, and the caps could be visualized for up to 30 min (Fig. 6). By contrast, the fluorescent circumference in mutant cells coalesced poorly into caps at any of the time points analyzed (Fig. 6). These results suggest that dynamic reorganization of the cytoskeleton that accompanies the cross-linking of surface IgM is impaired in Pfdn1-deficient B cells.

FIGURE 6.

Capping defect in Pfdn1-deficient B cells. A, Splenocytes from heterozygous control and Pfdn1-deficient mice were incubated with Cy5-fluorescent labeled anti-IgM F(ab′)2 Abs on ice and either left on the ice (time 0) or incubated at 37°C for the indicated times. Cells were cytospun, fixed, and analyzed as described in Materials and Methods. Only surface IgM+ B cells were analyzed. Polarization of cell surface IgM into a concentrated cap was noticeably slower and did not form a concentrated cap in Pfdn1-deficient B cells. Phase contrast image is shown under each sample to display all cells in the field of analysis. B, The frequency of B cells with capped BCR from two independent experiments are shown. At least 50 cells were scored at each time point.

FIGURE 6.

Capping defect in Pfdn1-deficient B cells. A, Splenocytes from heterozygous control and Pfdn1-deficient mice were incubated with Cy5-fluorescent labeled anti-IgM F(ab′)2 Abs on ice and either left on the ice (time 0) or incubated at 37°C for the indicated times. Cells were cytospun, fixed, and analyzed as described in Materials and Methods. Only surface IgM+ B cells were analyzed. Polarization of cell surface IgM into a concentrated cap was noticeably slower and did not form a concentrated cap in Pfdn1-deficient B cells. Phase contrast image is shown under each sample to display all cells in the field of analysis. B, The frequency of B cells with capped BCR from two independent experiments are shown. At least 50 cells were scored at each time point.

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Reorganization and/or aggregation of surface Ag receptors is an important component of T and B cell activation by specific Ags. We therefore asked whether the capping defects of Pfdn1-deficient B cells and actin polymerization defects in Pfdn1-deficient T cells (data not shown) were accompanied by activation defects following Ag receptor cross-linking. As shown in Fig. 7,A, CD19+IgD+ splenic B cells from wild-type mice were activated by either anti-IgM or PMA/ionomycin as assessed by expression of the activation marker CD86. By contrast, Pfdn1-deficient B cells were only weakly activated by anti-IgM (Fig. 7,B) despite the fact that the mutant cells expressed similar levels of cell surface IgM (Fig. 2,D) but were activated by PMA/ionomycin. Although less pronounced, Pfdn1-deficient T cells also displayed activation defects following treatment with anti-CD3 as assessed by CD69 expression (Fig. 7, C and D).

FIGURE 7.

Activation defect in Pfdn1-deficient B (A and B) and T cells (C and D). CD19+IgM+IgD+ spleen B cells from wild-type (A) and Pfdn1-deficient mice (B) were treated with anti-IgM F(ab′)2 (dark lines), PMA/ionomycin (dotted lines), or medium alone (gray lines), and after 16 h cell activation was assessed by flow cytometry. Pfdn1-deficient B cells were activated by PMA/ionomycin but not by anti-IgM as assessed by anti-CD86 staining. The mean fluorescent intensities (MFIs) of wild-type and mutant B cells before and after anti-IgM treatment were 95 and 339, whereas in mutant B cells the MFIs were 48 and 66, respectively. CD4+ T cells from wild-type (C) and Pfdn1-deficient mice (D) were treated with anti-CD3 (dark lines), PMA/ionomycin (dotted lines), or medium alone (gray lines), and after 16 h cell activation was assessed by flow cytometry. Pfdn1-deficient T cells were activated by PMA/ionomycin but not by anti-CD3 as assessed by anti-CD69 staining. The MFIs (CD69) of wild-type/mutant T cells before and after anti-CD3 treatment were 6 and 151, whereas MFI of mutant T cells were 5 and 28, respectively.

FIGURE 7.

Activation defect in Pfdn1-deficient B (A and B) and T cells (C and D). CD19+IgM+IgD+ spleen B cells from wild-type (A) and Pfdn1-deficient mice (B) were treated with anti-IgM F(ab′)2 (dark lines), PMA/ionomycin (dotted lines), or medium alone (gray lines), and after 16 h cell activation was assessed by flow cytometry. Pfdn1-deficient B cells were activated by PMA/ionomycin but not by anti-IgM as assessed by anti-CD86 staining. The mean fluorescent intensities (MFIs) of wild-type and mutant B cells before and after anti-IgM treatment were 95 and 339, whereas in mutant B cells the MFIs were 48 and 66, respectively. CD4+ T cells from wild-type (C) and Pfdn1-deficient mice (D) were treated with anti-CD3 (dark lines), PMA/ionomycin (dotted lines), or medium alone (gray lines), and after 16 h cell activation was assessed by flow cytometry. Pfdn1-deficient T cells were activated by PMA/ionomycin but not by anti-CD3 as assessed by anti-CD69 staining. The MFIs (CD69) of wild-type/mutant T cells before and after anti-CD3 treatment were 6 and 151, whereas MFI of mutant T cells were 5 and 28, respectively.

Close modal

The present study describes the first genetic analysis of mammalian Prefoldin, a conserved chaperone required for efficient folding of cytoskeletal subunit proteins. Pfdn1-deficient mice developed to term but displayed severe abnormalities in lymphocyte development and function, mucus clearance defects, hydrocephaly, and loss of nerve bundles in the CNS. Thus, mammalian Pfdn1, like the orthologous genes of Saccharomyces cerevisiae and C. elegans, encodes nonredundant functions that cannot be replaced by other chaperones. The requirement for Pfdn1 is also consistent with biochemical reconstitution experiments in which all six subunits are required for assembly and function of the Prefoldin complex (9, 10, 13).

Pfdn1 mutant phenotypes, although varied, were all consistent with abnormal cytoskeletal function. The defects in B and T cell development and function were similar to but more severe than mutations in proteins (WASP and WIP) that regulate actin polymerization in lymphocytes (5). Actin and tubulin cytoskeletons are prominent features of the neural growth cone and orchestrate axon outgrowth and guidance (4). Finally, hydrocephaly and respiratory mucus accumulation/infection are classic manifestations of immotile cilia syndrome (ciliary dyskinesia) caused by microtubule defects (28). This suggests that cytoskeletal proteins comprise the major substrates for Prefoldin function in mammals as in S. cerevisiae. However, the phenotypes of Pfdn1-deficient mice were more restricted than might be expected, considering the ubiquitous involvement of the cytoskeleton in diverse cellular processes (2, 3). Apparently, basic functions of the cytoskeleton in processes such as cell division, cell shape, and intracellular trafficking were not sufficiently impaired to affect the development of most tissues, at least as viewed on an anatomical level. For example, Prefoldin-deficient mice lacked neural tube closure defects often associated with genes involved in actin regulation and mitotic function (29). Because yeast Prefoldin enhances the rate of actin folding with little effect on steady-state protein levels (13), cellular processes that require rapid and extensive cytoskeletal synthesis or remodeling, such as immune cell signaling and axon guidance, may be more sensitive to the loss of Pfdn1 function.

Commissures consist of major tracts of neurons that connect regions of the CNS. Commissure defects frequently result from mutations in genes such as Enah (Mena) (30, 31), Mapk8 (Jnk1) (32) Nr2f1 (COUP-TFI) (33), Mtap1b (MAP1B) (34), and possibly Scar/WAVE1 (35, 36), which participate in neuron outgrowth/axon guidance by influencing cytoskeletal expression and function. These phenotypes illustrate the importance of the actin and tubulin cytoskeletons in the neural growth cone, which is present at the distal end of extending axons and orchestrates axon outgrowth and guidance (4). Similarly, the CNS phenotypes in Pfdn1-deficient mice are consistent with defects in actin and/or tubulin folding. Chaperone mutations can also cause neurodegenerative diseases due to the toxic effects of misfolded proteins (20, 22). Although neuronal phenotypes in Pfdn1-deficient mice could result from toxicity caused by aggregates of misfolded proteins, this seems less likely considering the overall pattern of neuronal cell loss.

Involvement of the actin cytoskeleton in lymphocyte function is illustrated by mutations in the genes encoding WASP (37, 38) and the WASP-interacting protein WIP (39). WASP belongs to a family of proteins (including N-WASP and Scar/WAVEs) that activate the actin-nucleating activity of the Arp2/3 (actin-related protein 2 and 3) complex. WASP, when recruited to an activated TCR, interacts with the Arp2/3 complex to promote cytoskeletal reorganization. Rapid actin polymerization provides a motile force that physically clusters TCRs into a synapse and generates a scaffold of actin filaments involved in sustained cell signaling (reviewed in Ref. 5). WASP expression is restricted to hematopoietic cells, thus limiting the phenotype of WASP knockout mice. WIP interacts with WASP in resting T cells, and the complex translocates to the immune synapse following TCR engagement. WIP-deficient T cells develop normally but display cell activation and actin assembly defects after TCR ligation and conjugate less efficiently with super Ag-presenting B cells (39).

Cell activation and Ag receptor capping defects in Pfdn1-deficient T and B cells recapitulate features of the WASP and WIP knockouts. However, the effects of the Pfdn1 mutation on lymphocyte development and function were more severe, as evidenced by developmental and functional defects affecting both T and B cells. The phenotype of Pfdn1-deficient mice is consistent with an actin-folding defect that could indirectly influence cytoskeleton assembly at a level upstream of WASP and WIP, although the overall consequences of Pfdn1 mutation were more restricted than those involving other regulators of actin assembly, including N-WASP (40, 41), Scar/WAVE2 (42, 43), and Nck1 together with Nck2 (44), because the latter impair embryonic development.

Lymphopoiesis defects in Pfdn1−/− mice included dramatic reductions in pre-B cells in the BM and in immature CD4+CD8+ double-positive T cells in the thymus. The eventual production of mature B and T cells suggests that Prefoldin is required primarily during the genesis and/or renewal of lymphocyte precursors rather than at later stages of lymphocyte development. These developmental defects precede the expression of functional Ag receptors and therefore differ from the activation defects observed in mature B and T cells. However, the defects in both B and T cell development occurred at stages that require cell migration and physical interactions with stromal cells in the microenvironment of the thymus and BM (45, 46). An additional developmental defect resulting from impaired BCR signaling is suggested by substantial increases in the proportion of MZ B cells relative to follicular B cells in Pfdn1 mutant mice, because the formation of MZ B cells is thought to require a lower threshold of Ag receptor signaling than mature follicular B cells (47). Alternatively, immune cell defects could be a secondary consequence of cellular or systemic stress in the mutant animals. Additional experiments will be required to clarify the roles of Prefoldin in early lymphocyte development and Ag receptor-dependent lymphocyte maturation.

Lymphocyte activation and actin assembly defects raise questions about the mechanism by which Pfdn1 mutation influences cytoskeletal function. The fact that Prefoldin (Gim) function is not essential for cell viability in S. cerevisiae (9, 10) has been attributed to the ability of substrates to fold at slower rates without functionally interacting with Prefoldin (13, 48). However, if Prefoldin simply enhances the rate of protein folding, then resting lymphocytes should contain adequate pools of properly folded proteins for processes, such as F-actin assembly and receptor capping, that occur within minutes after Ag receptor cross-linking. Indeed, wild-type and Pfdn1-deficient lymphocytes express similar levels of β-actin protein as assessed by Western blot analysis (data not shown), suggesting that the activation and actin assembly defects does not result from inadequate pools of cytoskeletal subunit proteins. In principle, Prefoldin could play other roles in cytoskeletal assembly and/or remodeling that are separate from cotranslational protein folding. In addition, Pfdn1-deficient cells could harbor sufficient levels of improperly folded subunits to poison polymer assembly upon cell activation. Although additional experiments will be required to examine these issues, the effects of Pfdn1 deficiency resemble aging-associated defects in lymphocyte development and function (49, 50). Considering that altered cellular proteins accumulate during aging and contribute to a number of chronic diseases (21, 22), the present study implicates misfolded cytoskeletal proteins as a cause of age-related defects in lymphocyte function.

We thank Abudi Nashabi for technical assistance, Drs. Danyvid Olivares-Villagomez and Luc Van Kaer for help with FACS, Dr. Robert Collins for histopathology analysis, and Dr. Emily Clark for expert analysis of BCR capped B cells.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by Public Health Service Grants P01HL68744 (to H.E.R.) and AI060729-01 (to W.N.K.). K.L.H. was supported by National Institutes of Health Grant F32-AI069770-01. Additional support was provided by the Department of Microbiology and Immunology of Vanderbilt University and by Cancer Center Support Grant P30CA68485 to the Vanderbilt-Ingram Cancer Center.

5

Abbreviations used in this paper: ES, embryonic stem; BM, bone marrow; MEF, mouse embryonic fibroblast; MZ, marginal zone; WASP, Wiskott-Aldrich syndrome protein; WIP, WASP-interacting protein; MFI, mean fluorescent intensity.

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