Cellular Mechanisms For Transferrin Receptor Endocytosis Vital For Terminal Erythroid Differentiation

Clathrin-mediated endocytosis (CME) is a key endocytic portal that regulates transferrin receptor (TfR) endocytosis. Although CME is among the most-studied cellular systems in cell biology, little is known regarding how this key system physiologically functions in vivo.The CALM gene (for Clathrin Assembly Lymphoid Myeloid, also known as PICALM), located on human chromosome 11q14, encodes a 652 aa protein containing multiple domains functioning in clathrin-coated vesicle formation. Since CALM is predominantly expressed in erythroblasts, and hematopoietic-specific Calmconditional knockout mice, which we developed, exhibit a severe iron-deficient anemia, we hypothesized that CALM functions to increase efficiency of TfR endocytosis in erythroblasts so that iron uptake is not rate-limiting. To test this hypothesis, we performed FACS-based endocytosis assay in primary erythroblasts, in vitro erythroid differentiation assay, live-cell imaging and CALM add-back rescue experiments.

TfR endocytosis was severely attenuated in Calm-deficient basophilic- and polychromatophilic erythroblasts: efficiency of TfR endocytosis was less than 25% of that of control, as revealed by FACS-based endocytosis assay. Unexpectedly, TfR endocytosis was not active in orthochromatophilic erythroblasts regardless of genotype, while they express TfR at high levels.For live cell imaging, we established immortalized mouse embryonic fibroblasts (MEFs), in which the Calm gene can be deleted in an inducible manner (Calm^F/F ERT2-Cre+ MEFs). The MEFs were then stably transduced with the retrovirus encoding an EGFP-tagged AP2-bound adaptin σ2. The dynamics of clathrin-coated vesicle formation was determined using fast live cell fluorescence spinning disk confocal microscopy. Clathrin-coat dynamics was barely affected in the absence of Calm under regular culture condition; however, it was completely stalled under high membrane tension (e.g. hypo-osmotic medium, jasplakinolide treatment). To determine functional domain(s) of CALM, a series of CALM mutants were generated, “added-back” to Calm-deficient MEFs and coat dynamics examined by live cell imaging. Mutant that does not bind to phosphatidylinositol 4,5-bisphosphate of the plasma membrane (PIP2-mutant CALM) failed to rescue TfR endocytosis in Calm-deficient MEFs, suggesting that CALM binding to PIP₂, but not to v-SNAREs or EPS15, is essential for CALM-mediated TfR endocytosis.

Splenic erythroblasts were significantly expanded in Calm conditional knockout mice. In contrast, Calm-deficient hematopoietic progenitors cannot give rise to immature erythroblasts in vitro, suggesting that Calm-deficiency is partially compensated in vivo via non-cell autonomous mechanisms (e.g. central macrophages). Iron supplement treatment could significantly rescue erythroid development of Calm-deficient progenitors in vitro, indicating that iron-deficiency is a primary cause of developmental defects seen in Calm-deficient erythroblasts. As expected, retrovirus-mediated expression of WT-Calm, but not PIP₂-mutant, completely rescued erythroid development in Calm-deficient erythroblasts in vitro.Our data indicate that CALM plays a key role in clathrin-mediated endocytosis in a context-dependent (high membrane tension) and cell-type-specific (erythroblasts) manner. We propose that CALM is essential for transferrin uptake in erythroblasts by functioning as an erythroid-specific clathrin adaptor.