17Coordinated assembly and disassembly of integrin-mediated focal adhesions (FAs) is essential for cell migration. 18 Many studies have shown that FA disassembly requires Ca 2+ influx, however our understanding of this process 19 remains incomplete. Here we show that Ca 2+ influx via STIM1/Orai1 calcium channels, which cluster near FAs, 20 leads to activation of the GTPase Arf5 via the Ca 2+ -activated GEF IQSec1, and that both IQSec1 and Arf5 21 activation are essential for adhesion disassembly. We further show that IQSec1 forms a complex with the lipid 22 transfer protein ORP3, and that Ca 2+ influx triggers PKC-dependent translocation of this complex to ER/plasma 23 membrane contact sites adjacent to FAs. In addition to allosterically activating IQSec1, ORP3 also extracts PI4P 24 from the PM, in exchange for phosphatidylcholine. ORP3-mediated lipid exchange is also important for FA 25 turnover. Together, these findings identify a new pathway that links calcium influx to FA turnover during cell 26 migration. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 100 activation of Arf5 and insertion of PC into the plasma membrane are both important to promote adhesion 101disassembly. Taken together, our findings establish a spatial and mechanistic link between calcium influx at 102 ER/PM contact sites and focal adhesion turnover mediated by ORP3 and IQSec1-activated Arf5.

Coordinated assembly and disassembly of integrin-mediated focal adhesions (FAs) is essential for cell migration. Many studies have shown that FA disassembly requires Ca2+ influx, however our understanding of this process remains incomplete. Here, we show that Ca2+ influx via STIM1/Orai1 calcium channels, which cluster near FAs, leads to activation of the GTPase Arf5 via the Ca2+-activated GEF IQSec1, and that both IQSec1 and Arf5 activation are essential for adhesion disassembly. We further show that IQSec1 forms a complex with the lipid transfer protein ORP3, and that Ca2+ influx triggers PKC-dependent translocation of this complex to ER/plasma membrane (PM) contact sites adjacent to FAs. In addition to allosterically activating IQSec1, ORP3 also extracts PI4P from the PM, in exchange for phosphatidylcholine. ORP3-mediated lipid exchange is also important for FA turnover. Together, these findings identify a new pathway that links calcium influx to FA turnover during cell migration.

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BACKGROUND: Measurement of abnormal Red Blood Cell (RBC) deformability is a main indicator of Sickle Cell Anemia (SCA) and requires standardized quantification methods. Ektacytometry is commonly used to estimate the fraction of Sickled Cells (SCs) by measuring the deformability of RBCs from laser diffraction patterns under varying shear stress. In addition to estimations from model comparisons, use of maximum Elongation Index differences (ΔEImax) at different laser intensity levels was recently proposed for the estimation of SC fractions. OBJECTIVE: Implement a convolutional neural network to accurately estimate rigid-cell fraction and RBC concentration from laser diffraction patterns without using a theoretical model and eliminating the ektacytometer dependency for deformability measurements. METHODS: RBCs were collected from control patients. Rigid-cell fraction experiments were performed using varying concentrations of glutaraldehyde. Serial dilutions were used for varying the concentration of RBC. A convolutional neural network was constructed using Python and TensorFlow. RESULTS: Our measurements and model predictions show that a linear relationship between ΔEImax and rigid-cell fraction exists only for rigid-cell fractions less than 0.2. Our proposed neural network architecture can be used successfully for both RBC concentration and rigid-cell fraction estimations without a need for a theoretical model.

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