The PICALM protein plays a key role in iron homeostasis and cell proliferation

Abstract

The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimer's disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM's function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.

Figure 1. The carboxy-terminal 69 amino acids of PICALM are essential for binding clathrin and interfering with TfR endocytosis.

(A) Schematic representation of amino-terminal-tagged GFP-PICALM fusion proteins. (B) TfR internalization in HEK293 cells transiently transfected with empty vector (control), full length PICALM (PICALM: 1–652), amino-terminal truncated PICALM (PICALM: 256–652), or a PICALM mutant missing both the amino and carboxy-terminal regions (PICALM: 256–583). The relative rate of endocytosis was calculated as described in Materials and Methods. N_exp  = 11. *p<0.001 compared with control. (C) Western blot of proteins co-immunoprecipitated (IP) with anti-GFP antibody and immunoblotted (IB) with anti-clathrin heavy chain (CHC) antibody (upper panel). Cell lysates (input) were also immunoblotted with anti-CHC and anti-PICALM antibodies (lower panels). The molecular weights of GFP-PICALM, GFP-PICALM:256–652 and GFP-PICALM:256–583 are 97kD, 70kD and 62 kD, respectively.

Figure 2. Absence of PICALM results in a decreased rate of internalization and increased cell surface expression of TfR in MEFs.

The level of cell surface TfR expression normalized to levels in WT cells (A,C,E) and the rate of TfR internalization (B,D,F) was measured in non-immortalized MEFs (A,B), immortalized MEFs (C,D), or PICALM shRNA knockdown WT (line 7T) MEFs (E,F). (A) Surface TfR expression in non-immortalized WT and Picalm ^NULL MEFs. N_exp  = 4. *p<0.02 compared with WT. (B) TfR internalization at three different time points in independently derived non-immortalized WT (lines 20, 33) and Picalm ^NULL (lines 24, 29, 31). N_exp  = 2. *p<0.04 compared with WT. (C) Surface TfR expression in immortalized WT and Picalm ^NULL MEFs. N_exp  = 4. *p<0.04 compared with WT. (D) TfR internalization in independently derived immortalized WT (lines 20T, 33T) and Picalm ^NULL (lines 24T, 29T, 31T). N_exp  = 7. *p<0.01 compared with WT. (E) Surface TfR expression in shRNA transduced WT MEFs. N_exp  = 4. *p<0.02 compared with WT. (F) TfR internalization in immortalized WT (line 7T) transduced with two separate PICALM shRNA retroviruses (shRNA4, shRNA5) or a control shRNA retrovirus. N_exp  = 3. *p<0.01 compared with WT. In all cases, error bars denote standard error. Two-tailed Student’s t-test was used to compare means for TfR internalization (A,C,E).

Figure 3. PICALM-deficient cells display increased total TfR protein and mRNA.

(A) Immunoblot of total TfR protein, PICALM, and β-actin from Picalm ^NULL (Null), WT, Picalm ^NULL control MEFs infected with empty vector (Null + control), or Picalm ^NULL MEFs rescued with PICALM (Null + PICALM). PICALM cDNA encodes for the larger isoform. (B) Quantitation of TfR shown in panel A immunoblot normalized to β-actin, with values shown relative to TfR level in WT MEFs. N_exp  = 3. *p<0.04. (C) RT-PCR quantitation of TfR mRNA in PICALM-deficient (Null) MEFs, WT MEFs, Picalm ^NULL MEFs transduced with empty vector (Null + Control) or rescued with PICALM (Null + PICALM). Results are normalized to levels in Null MEFs or Null + Control cells. N_exp  = 3. *p<0.04 compared with WT cells.

Figure 4. PICALM expression rescues the PICALM-deficient phenotype, and the PICALM carboxy-terminal domain plays an essential role.

(A) Representative experiment showing rate of TfR internalization in Picalm ^NULL MEFs (line 31T) stably infected with PICALM or empty vector (control) compared with WT (line 33T) MEFs. (B) Surface TfR expression in WT MEFs (7T, 20T, 33T) or Picalm ^NULL MEFs (lines 3T, 24T, 29T, 31T) that were uninfected, stably infected with empty vector (control) or PICALM. Surface TfR levels were normalized to levels in control Picalm ^NULL MEFs. N_exp  = 8. *p<0.001 compared with control. TfR endocytosis and surface TfR levels measured in three independent Picalm ^NULL lines is shown in Figure S3. (C) Deletion mutagenesis of the PICALM carboxy-terminal domain (constructs depicted in left panel) demonstrates that the ability to rescue surface TfR expression is dependent on the PICALM carboxy-terminal domain. N_exp  = 3. *p<0.02 compared with PICALM-17a. (D) Co-immunoprecipitation of lysates from HEK293 cells transiently transfected with full length PICALM (PICALM: 1–652), carboxy-terminal truncated PICALM (PICALM: 1–583), or a PICALM mutant missing both the amino and carboxy-terminal regions (PICALM: 256–583). Extracts were immunoprecipitated (IP) with anti-GFP antibody and immunoblotted (IB) with anti-clathrin heavy chain (CHC) antibody (upper panel). Cell lysates (input) were also immunoblotted with anti-PICALM antibodies (lower panel).

Figure 5. PICALM-deficient MEFs exhibit decreased intracellular iron compared with WT or PICALM rescued MEFs.

(A) Chelation of Phen Green SK (the fluorescence of which inversely correlates with intracellular iron levels) was measured in WT (lines 7T, 20T) or PICALM-deficient (Picalm ^NULL – lines 3T, 24T, 29T) MEFs. Values were normalized to those in WT MEFs. N_exp  = 3. *p<0.04 compared with WT. (B) Chelation of Phen Green SK in Picalm ^NULL cells that were uninfected or rescued with PICALM. Values are normalized to levels in PICALM-rescued cells. N_exp  = 3. *p<0.04 compared with PICALM-rescued cells. (C) Total intracellular iron levels in Picalm ^NULL (line 3T) and PICALM rescued MEFs, normalized to levels in Picalm ^NULL cells. N_exp  = 3. MFI: mean fluorescence intensity.

Figure 6. PICALM-deficient MEFs proliferate more rapidly in the presence of iron supplementation and are more sensitive to iron chelation.

(A,B) Representative proliferation curves of non-immortalized Picalm ^NULL and WT MEFs without (A) and with (B) 50 µM ferric ammonium citrate (FAC). FAC restores proliferation in Picalm ^NULL cells to levels in WT cells. (C,D) Mean cell numbers (+/− standard error) of non-immortalized Picalm ^NULL and WT MEFs grown for 5 days without (C) and with (D) 50 µM FAC. N_exp  = 3. *p<0.04 compared with WT cells. (E,F) Representative proliferation curves of immortalized Picalm ^NULL MEFs transduced with empty vector (control) or PICALM grown for 4 days in the absence (E) or presence (F) of 2.5 µM deferoxamine (DFO). (G) Mean cell number of Picalm ^NULL MEFs untransduced (Null 3T) or transduced with empty vector (control) or PICALM after 4 days of culture in the presence of 2.5 µM DFO treatment relative to number of untreated cells. N_exp  = 4. *p<0.003 compared with PICALM rescued cells.