Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein ​CrkL, a major substrate of the oncogenic tyrosine kinase ​BCR-​ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <104 cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-​CrkL and the protein tyrosine phosphatase ​PTPRC/​CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents

FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326–380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the “Claw” for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation

Cell Sorting in Pillar Arrays based on Electrokinetics and Morphology

Deterministic Lateral Displacement (DLD) is a method capable of sorting cells based on size where mechanicalinteractions between a sufficiently large particle and obstacles in a microfludic pillar array force the particle tofollow a different trajectory than their smaller counterparts, resulting in continuous lateral separation. To extendthe capability of DLD, electrical interaction between particles and pillars can be employed to complement themechanical interaction, making electrical/dielectric properties additional parameters for sorting. Another idea isto exploit the morphologies of cells and as a concequence, their dynamical properties, to sort them in DLD. Thedevelopment of DLD cell sorting methods based on those two ideas has brought forth five papers appended to thisthesis: paper I, III, and V (combination of electrokinetics and DLD), and paper II and IV (exploiting morphologyin sorting by DLD).In the first topic, differences in electric properties or dielectric properties of particles and cells are employed toextend the capability of DLD. In Paper I, an AC electric field was applied across DLD devices having insulatingpillars to sort similar-sized polystyrene particles having different surface charge, viable from non-viable yeast cells,and viable from non-viable E. coli bacteria. In Paper III, the same method was utilised on open channel DLDdevices, showing unaltered effectiveness but offering the ability to flexibly change the distance between the electrodes.Also in the topic of combining electrokinetics and DLD, Paper V introduced a new type of DLD devicewhere the electrodes were defined locally on every pillar, making it easier to generate a high electric field strength.Besides electrical properties, morphology is another useful accompaniment to DLD. In Paper II, pathogenicStreptococcus pneumoniae bacteria were fractionated in DLD devices according to the difference in their morphology,viz. their chain length. It was also demonstrated, in paper IV, that an AC field can be used to rotatenon-spherical red blood cells and in turn, change their trajectory in a DLD device. This implies an opportunity tosort red blood cells from cells having different morphology, either spherical cells or parasites like trypanosomes

Expression and structure-functional studies of human apolipoproteinciii

Apolipoprotein (Apo) CIII plays a key role in triglyceride (TG)-rich lipoprotein metabolism and is a risk factor for coronary heart disease (CHD). The study involved in this thesis is the first in vitro structure-functional study using recombinant apoCIII proteins. The physicochemical properties of recombinant wild type and A23T, a naturally occurring mutation that is associated with apoCIII deficiency and lower plasma TG levels, as well as three site-directed mutants of apoCIII, designed by molecular modelling and implicated in lipid binding (L9T/T20L, F64A/W65A) or lipoprotein lipase (LPL) inhibition (K21A), were compared. Relative lipid binding efficiencies of each apoCIII variants to 1.2-dimyristoyl-sn-glycero-3- phosphatidylcholine (DMPC) were: L9T/T20L>WT>K21A>A23T>F64A/W65A with an inverse correlation with size of the discoidal complexes formed. Physicochemical analysis (Trp fluorescence, circular dichroism (CD) and GdnHC1 denaturation) suggested that the stability of the resulting apoCIII:DMPC complexes were dependent on their lipid binding properties. The displacement of apoE by apoCIII variants were tested by gel filtration of apoE:dipalmitoylphosphatidylcholine (DPPC) discoidal complexes mixed with the various apoCIII variants. All apoCIII proteins bound to the apoE:DPPC complexes and the capacity to displace apoE from the complex was dependent on their lipid binding affinity. All the recombinant apoCIII proteins inhibited LPL in the presence or in the absence of apoCII, with F64A/W65A displaying the most inhibition, suggesting that apoCIII inhibition of LPL is independent of lipid binding and therefore due to the protein:protein interaction with apoCII and/or LPL. Taken together, our data suggest that the hydrophobic residues F64 and W65 are crucial for the lipid binding properties of apoCIII and the redistribution of the N- terminal helix of apoCIII (L9T/T20L permutation) can enhance the lipid binding properties of the protein. Additionally, the reduced lipid binding capacity of the naturally occurring mutation A23T could lead to reduced plasma apoCIII and lower plasma TG levels in carriers