Hector H. Huang

Monitoring Lipid Anchor Organization in Cell Membranes by PIE-FCCS

This study examines the dynamic co-localization of lipid-anchored fluorescent proteins in living cells using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) and fluorescence lifetime analysis. Specifically, we look at the pairwise co-localization of anchors from lymphocyte cell kinase (LCK: myristoyl, palmitoyl, palmitoyl), RhoA (geranylgeranyl), and K-Ras (farnesyl) proteins in different cell types. In Jurkat cells, a density-dependent increase in cross-correlation among RhoA anchors is observed, while LCK anchors exhibit a more moderate increase and broader distribution. No correlation was detected among K-Ras anchors or between any of the different anchor types studied. Fluorescence lifetime data reveal no significant Förster resonance energy transfer in any of the data. In COS 7 cells, minimal correlation was detected among LCK or RhoA anchors. Taken together, these observations suggest that some lipid anchors take part in anchor-specific co-clustering with other existing clusters of native proteins and lipids in the membrane. Importantly, these observations do not support a simple interpretation of lipid anchor-mediated organization driven by partitioning based on binary lipid phase separation.

Live Cell Plasma Membranes Do Not Exhibit a Miscibility Phase Transition over a Wide Range of Temperatures

Lipid/cholesterol mixtures derived from cell membranes as well as their synthetic reconstitutions exhibit well-defined miscibility phase transitions and critical phenomena near physiological temperatures. This suggests that lipid/cholesterol-mediated phase separation plays a role in the organization of live cell membranes. However, macroscopic lipid-phase separation is not generally observed in cell membranes, and the degree to which properties of isolated lipid mixtures are preserved in the cell membrane remain unknown. A fundamental property of phase transitions is that the variation of tagged particle diffusion with temperature exhibits an abrupt change as the system passes through the transition, even when the two phases are distributed in a nanometer-scale emulsion. We support this using a variety of Monte Carlo and atomistic simulations on model lipid membrane systems. However, temperature-dependent fluorescence correlation spectroscopy of labeled lipids and membrane-anchored proteins in live cell membranes shows a consistently smooth increase in the diffusion coefficient as a function of temperature. We find no evidence of a discrete miscibility phase transition throughout a wide range of temperatures: 14-37 °C. This contrasts the behavior of giant plasma membrane vesicles (GPMVs) blebbed from the same cells, which do exhibit phase transitions and macroscopic phase separation. Fluorescence lifetime analysis of a DiI probe in both cases reveals a significant environmental difference between the live cell and the GPMV. Taken together, these data suggest the live cell membrane may avoid the miscibility phase transition inherent to its lipid constituents by actively regulating physical parameters, such as tension, in the membrane.

Time-Resolved Proteomics Extends Ribosome Profiling-Based Measurements of Protein Synthesis Dynamics.

Ribosome profiling is a widespread tool for studying translational dynamics in human cells. Its central assumption is that ribosome footprint density on a transcript quantitatively reflects protein synthesis. Here, we test this assumption using pulsed-SILAC (pSILAC) high-accuracy targeted proteomics. We focus on multiple myeloma cells exposed to bortezomib, a first-line chemotherapy and proteasome inhibitor. In the absence of drug effects, we found that direct measurement of protein synthesis by pSILAC correlated well with indirect measurement of synthesis from ribosome footprint density. This correlation, however, broke down under bortezomib-induced stress. By developing a statistical model integrating longitudinal proteomic and mRNA-sequencing measurements, we found that proteomics could directly detect global alterations in translational rate caused by bortezomib; these changes are not detectable by ribosomal profiling alone. Further, by incorporating pSILAC data into a gene expression model, we predict cell-stress specific proteome remodeling events. These results demonstrate that pSILAC provides an important complement to ribosome profiling in measuring proteome dynamics.

Dynamic Organization of Myristoylated Src in the Live Cell Plasma Membrane.

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell-cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10-80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Taken together, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.

Time-resolved proteomics vs. ribosome profiling reveals translation dynamics under stress

Many small molecule chemotherapeutics induce stresses that globally inhibit mRNA translation, remodeling the cancer proteome and governing response to treatment. Here we measured protein synthesis in multiple myeloma cells treated with low-dose bortezomib by coupling pulsed-SILAC (pSILAC) with high-accuracy targeted quantitative proteomics. We found that direct measurement of protein synthesis by pSILAC correlated well with the indirect measurement of protein synthesis by ribosome profiling under conditions of robust translation. By developing a statistical model integrating longitudinal proteomic and mRNA-seq measurements, we found that proteomics could directly detect global alterations in translational rate as a function of therapy-induced stress after prolonged bortezomib exposure. Finally, the model we develop here, in combination with our experimental data including both protein synthesis and degradation, predicts changes in proteome remodeling under a variety of cellular perturbations. pSILAC therefore provides an important complement to ribosome profiling in directly measuring proteome dynamics under conditions of cellular stress.

EphA2-Ephrina1 Signaling and PI(4,5)P2 Spatial Organization on Breast Cancer Cells

EphA2 belongs to the largest subfamily - Eph receptors - of the Receptor Tyrosine Kinase (RTK) superfamily and is over-expressed in many cancer cell lines. The major role of Eph receptors is to regulate the dynamics of cellular protrusions and cell migration. Previous research reports that activation of EphA2 by its ligand ephrinA1 increases the activity of Phosphoinositide 3-kinase (PI3K). PI3K is one of the key molecules in regulating cell migration by phosphorylating Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) to Phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) at the cell edge facing the highest chemoattractant concentration.Here, we recapitulate EphA2-EphrinA1 signaling between cells by presenting breast cancer cells expressing EphA2 with an ephrinA1-displaying supported lipid bilayer. Through live cell labeling of PI(4,5)P2 with the fluorescent PLCδ1-PH domain biosensor, we are able to directly monitor PI(4,5)P2 spatial organization and its role in EphA2 signaling pathway. In addition, PI(4,5)P2 signaling and membrane localization are also examined with a spatial mutation strategy, which presents diffusion barriers, disrupting EphA2-ephrinA1 spatial organization. Our study will further clarify the role of PI(4,5)P2 and PI3K in the EphA2 signaling pathway, and help to understand cancer cell progression and metastasis in the long term.

Concentration Dependent Membrane Anchor Colocalization Study by Fluorescence Cross-Correlation Spectroscopy in Live Cells

Membrane anchors such as protein lipidations and glypiations have been proposed to play essential roles in the sorting and organization of plasma membrane-associated proteins, especially those involved in cell signaling. Here, we investigate the concentration dependence and variability of anchor colocalization in live cells by transfecting various cell types with pairs of fusion proteins created by replacing all but short tails of natively lipidated proteins with either red or green fluorescent proteins. These fusion proteins remove any native protein-protein interactions while fluorescently tagging membrane anchors in live cells. To observe sub-cellular organization, we use Fluorescence Cross-Correlation Spectroscopy (FCCS) to quantify the dynamic colocalization between green- and red-labeled anchors. FCCS allows observations of dynamic colocalization in live cells at a greater range of separation distances than is allowed by FRET, and because it is a dynamic measurement FCCS avoids ambiguous or false positive colocalization that can result from static studies. Fusion protein expression level, as determined by overall intensity of cell fluorescence, naturally varies in a population of transiently transfected cells. Using this to our advantage, we are able to observe cells within a wide range of protein expression and explore trends between concentration and fusion protein colocalization. We also analyze variation in the amount of colocalization and observe a difference between the variability from cell-to-cell and the variability from spot-to-spot within one cell across several anchor types and different cell lines.

Trapping and Polymerization of Tubulin Within Phospholipid Vesicles

Microtubules are cylindrical aggregates (diameter ∼25 nm) of the protein tubulin. They play important roles in cellular structure, protein trafficking, cell motility, intracellular transport, meiosis, and mitosis. They can switch stochastically between periods of tubulin polymerization and dissolution, termed dynamic instability, and can individually apply an extensive force at a significant distance on the cellular scale (Howard and Hyman 2003). Previous light microscopy has been used to characterize the forces that produce membrane distortions when microtubules assemble inside lipid vesicles (Fygenson et al. 1997; Emsellem et al. 1998).We have examined this phenomenon by cryo-EM to gain insights on membrane deformation and to visualize the detailed structure of the microtubule ends as they are constrained near the inner membrane surface. Vesicles need to be of sufficient size, typically 1 µm diameter, to contain sufficient tubulin to form a microtubule long enough to span the vesicle. This has presented a challenge given limitations on the thickness of EM samples.To date, we have visualized microtubule polymers in even small vesicles that often produce significant distortion, along with microtubules that form outside the vesicles. Quite unexpectedly, it appears that in certain cases the vesicles are deformed by the microtubule, sometimes with long protrusions, while in other cases in vesicles of similar size, the microtubules are deformed by the vesicles, with a number of sharp bends. This presents an opportunity to gain insights on the factors involved in both lipid bilayer and microtubule strength and flexibility. Ultimately we will image these samples using electron tomography to describe all of the effects in three dimensions. Determination of the nature of the dynamic instability and microtubule growth and vesicle tensile strength resulting from microtubule elongation will provide insight into the nature of cellular architecture, plasticity, and deformation.