3D-STED Super-Resolution Microscopy Reveals Distinct Nanoscale Organization of the Hematopoietic Cell-Specific Lyn Substrate-1 (HS1) in Normal and Leukemic B Cells.

Marta Sampietro,Federica Barbaglio,Lydia Scarfò,Moreno Zamai,Cristina Scielzo,Valeria R Caiolfa,Veronica Labrador Cantarero,Alfonsa Díaz Torres

doi:10.3389/fcell.2021.655773

Abstract

HS1, the hematopoietic homolog of cortactin, acts as a versatile actin-binding protein in leucocytes. After phosphorylation, it is involved in GTPase and integrin activation, and in BCR, TCR, and CXCR4 downstream signaling. In normal and leukemic B cells, HS1 is a central cytoskeletal interactor and its phosphorylation and expression are prognostic factors in chronic lymphocytic leukemia (CLL) patients. We here introduce for the first time a super-resolution imaging study based on single-cell 3D-STED microscopy optimized for revealing and comparing the nanoscale distribution of endogenous HS1 in healthy B and CLL primary cells. Our study reveals that the endogenous HS1 forms heterogeneous nanoclusters, similar to those of YFP-HS1 overexpressed in the leukemic MEC1 cell line. HS1 nanoclusters in healthy and leukemic B cells form bulky assemblies at the basal sides, suggesting the recruitment of HS1 for cell adhesion. This observation agrees with a phasor-FLIM-FRET and STED colocalization analyses of the endogenous MEC1-HS1, indicating an increased interaction with Vimentin at the cell adhesion sites. In CLL cells isolated from patients with poor prognosis, we observed a larger accumulation of HS1 at the basal region and a higher density of HS1 nanoclusters in the central regions of the cells if compared to good-prognosis CLL and healthy B cells, suggesting a different role for the protein in the cell types analyzed. Our 3D-STED approach lays the ground for revealing tiny differences of HS1 distribution, its functionally active forms, and colocalization with protein partners.

Highlights

The hematopoietic cell-specific lyn substrate-1 (HS1) protein is the hematopoietic homologue of cortactin (Schnoor et al, 2018), belonging to the class II nucleation-promoting factor (NPF) family able to initiate branched actin network assembly by activating the Arp2/3 complex (Helgeson and Nolen, 2013).The multi-domain sequence of HS1 is responsible for the recruitment of the protein in different mechanisms such as the activation of GTPases and integrins and the downstream signaling of BCR and TCR, and is indispensable for signaling events leading to actin assembly during immunological synapse (IS) formation
To evaluate possible differences in HS1 activity linked to its localization and intracellular nanostructure in chronic lymphocytic leukemia (CLL), we stratified the patients based on the mutational status of the immunoglobulin heavy variable gene (IGHV) being HS1 downstream the B cell receptor (CastroOchoa et al, 2019)
CLL clinical course reflects biological heterogeneity, and patients are usually classified in two main subsets depending on good or poor prognosis, which are based on a set of prognostic factors for the disease outcome and survival

Summary

Introduction

The hematopoietic cell-specific lyn substrate-1 (HS1) protein is the hematopoietic homologue of cortactin (Schnoor et al, 2018), belonging to the class II nucleation-promoting factor (NPF) family able to initiate branched actin network assembly by activating the Arp2/3 complex (Helgeson and Nolen, 2013). The multi-domain sequence of HS1 is responsible for the recruitment of the protein in different mechanisms such as the activation of GTPases and integrins and the downstream signaling of BCR and TCR, and is indispensable for signaling events leading to actin assembly during immunological synapse (IS) formation (reviewed in Gomez et al, 2006; Castro-Ochoa et al, 2019). Upon BCR-induced cross-linking and Syk activation, HS1 is localized in membrane lipid rafts, suggesting an adapter role in the recruitment and assembly of actin (Hao et al, 2004). In T cells, phosphorylation links HS1 to multiple signaling proteins, including Lck, PLCγ1, and Vav, and is essential for the stable recruitment of Vav to the IS (Gomez et al, 2006)

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