An Optical Tweezers-Based Single-Cell Manipulation and Detection Platform for Probing Real-Time Cancer Cell Chemotaxis and Response to Tyrosine Kinase Inhibitor PD153035

We presented an approach to address cancer cell chemotaxis and response to tyrosine kinase inhibitor PD153035 at the single-cell level. We applied an optical tweezer system together with the platform at the single-cell level to manipulate an epidermal growth factor (EGF)-coated bead positioned close to the filopodia to locally stimulate HT29 cells, the human colon cancer cell line overexpressing the EGF receptor (EGFR). To address cancer cell chemotaxis, a single-cell movement model was also proposed to quantify the propagation speed at the leading and trailing edges of the cell along the chemosensing axis. This study focused on three perspectives: probing the chemosensing process mediated by EGF/EGFR signaling, investigating the mode of locomotion during the EGF-coated bead stimulation, and quantifying the effect of PD153035 on the EGF–EGFR transport pathway. The results showed that the filopodial actin filament is a sensory system for EGF detection. In addition, HT29 cells may use the filopodial actin filament to distinguish the presence or absence of the chemoattractant EGF. Furthermore, we demonstrated the high selectivity of PD153035 for EGFR and the reversibility of binding to EGFR. We anticipate that the proposed single-cell method could be applied to construct a rapid screening method for the detection and therapeutic evaluation of many types of cancer during chemotaxis.

Structural basis of adhesive binding by desmocollins and desmogleins

Desmosomes are intercellular adhesive junctions that impart strength to vertebrate tissues. Their dense, ordered intercellular attachments are formed by desmogleins (Dsgs) and desmocollins (Dscs), but the nature of trans-cellular interactions between these specialized cadherins is unclear. Here, using solution biophysics and coated-bead aggregation experiments, we demonstrate family-wise heterophilic specificity: All Dsgs form adhesive dimers with all Dscs, with affinities characteristic of each Dsg:Dsc pair. Crystal structures of ectodomains from Dsg2 and Dsg3 and from Dsc1 and Dsc2 show binding through a strand-swap mechanism similar to that of homophilic classical cadherins. However, conserved charged amino acids inhibit Dsg:Dsg and Dsc:Dsc interactions by same-charge repulsion and promote heterophilic Dsg:Dsc interactions through opposite-charge attraction. These findings show that Dsg:Dsc heterodimers represent the fundamental adhesive unit of desmosomes and provide a structural framework for understanding desmosome assembly.

EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin

ABSTRACTObligate intracellular bacteria, such asEhrlichia chaffeensis, perish unless they can enter eukaryotic cells.E. chaffeensisis the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease. To infect cells,Ehrlichiauses theCterminus of the outer membrane invasinentry-triggeringprotein (EtpE) ofEhrlichia(EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl inositol-anchored protein, DNase X. How this binding drivesEhrlichiaentry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human proteins that interact with EtpE-C: CD147 and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibitedEhrlichiainternalization and infection but not binding. Downregulation of CD147 by short hairpin RNA (shRNA) impairedE. chaffeensisinfection. Functional ablation of cytoplasmic hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and infection but not binding. EtpE-C also interacted with neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by hnRNP-K. Wiskostatin, which inhibits N-WASP activation, and cytochalasin D, which inhibits actin polymerization, inhibitedEhrlichiaentry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulatedin vitroactin polymerization in an N-WASP- and DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to DNase X drivesEhrlichiaentry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization.IMPORTANCEEhrlichia chaffeensis, an obligate intracellular bacterium, causes a blood-borne disease called human monocytic ehrlichiosis, one of the most prevalent life-threatening emerging tick-transmitted infectious diseases in the United States. The survival ofEhrlichiabacteria, and hence, their ability to cause disease, depends on their specific mode of entry into eukaryotic host cells. Understanding the mechanism by whichE. chaffeensisenters cells will create new opportunities for developing effective therapies to prevent bacterial entry and disease in humans. Our findings reveal a novel cellular signaling pathway triggered by an ehrlichial surface protein called EtpE to induce its infectious entry. The results are also important from the viewpoint of human cell physiology because three EtpE-interacting human proteins, DNase X, CD147, and hnRNP-K, are hitherto unknown partners that drive the uptake of small particles, including bacteria, into human cells.