Biology subjects

Cupo, C.

Publications and source records attributed to Cupo, C..

3 recordsLinked to original sources

bioRxiv2021

Hyperspectral Counting of Multiplexed Nanoparticle Emitters in Single Cells and Organelles

Nanomaterials are the subject of a range of biomedical, commercial, and environmental investigations involving measurements in living cells and tissues. Accurate quantification of nanomaterials, at the tissue, cell, and organelle levels, is often difficult, however, in part due to their inhomogeneity. Here, we propose a method that uses the diverse optical properties of a nanomaterial preparation in order to improve quantification at the single-cell and organelle level. We developed hyperspectral counting, which employs diffraction-limited imaging via hyperspectral microscopy of a diverse set of nanomaterial emitters, to estimate nanomaterial counts in live cells and sub-cellular structures. A mathematical model was developed, and Monte Carlo simulations were employed, to improve the accuracy of these estimates, enabling quantification with single-cell and single-endosome resolution. We applied this nanometrology technique to identify an upper-limit of the rate of uptake into cells - approximately 3,000 particles endocytosed within 30 minutes. In contrast, conventional ROI counting results in a 230% undercount. The method identified significant heterogeneity and a broad non-Gaussian distribution of carbon nanotube uptake within cells. For example, while a particular cell contained an average of 1 nanotube per endosome, the heterogenous distribution resulted in over 7 nanotubes localizing within some endosomes, substantially changing the accounting of subcellular nanoparticle concentration distributions. This work presents a method to quantify cellular and subcellular concentrations of a heterogeneous carbon nanotube reference material, with implications for nanotoxicology, drug/gene delivery, and nanosensor fields.

bioengineering↗

bioRxiv2021

Optogenetic dissection of the roles of actomyosin in the mechanics underlying tissue fluidity

Rapid epithelial tissue flows are essential to building and shaping developing embryos. However, it is not well understood how the mechanical properties of tissues and the forces driving them to flow are jointly regulated to accommodate rapid tissue remodeling. To dissect the roles of actomyosin in the mechanics of epithelial tissue flows, here we use two optogenetic tools, optoGEF and optoGAP, to manipulate Rho/Rho-kinase signaling and actomyosin contractility in the germband epithelium, which flows via convergent extension movements during Drosophila body axis elongation. The ability to perturb actomyosin in the tissue allows us to analyze the effects of actomyosin on cell rearrangements, tissue tensions, and tissue mechanical properties. We find that either optogenetic activation or deactivation of Rho1 signaling and actomyosin contractility at the apical surface of the germband disrupts cell rearrangements and tissue-level flows. Rho1 activation leads to poorly oriented rearrangements that are associated with a redistribution of myosin II from the junctional to the medial-apical domain, whereas Rho1 deactivation leads to fewer, slower cell rearrangements that are associated with decreased junctional and medial myosin. By probing mechanical tensions in the tissue using laser ablation and inferring tissue mechanical properties from cell packings, we find that actomyosin influences both the anisotropic forces that drive tissue flow and the mechanical properties of the tissue that resist flow, leading to complex relationships between actomyosin activity and tissue fluidity. Moreover, our results link the subcellular distribution of myosin II to tissue tension and cell packings, revealing how junctional and medial myosin have differential roles in promoting and orienting cell rearrangements during tissue flow.

developmental biology↗

bioRxiv2021

Using optogenetics to link myosin patterns to contractile cell behaviors during convergent extension

Distinct spatiotemporal patterns of actomyosin contractility are often associated with particular epithelial tissue shape changes during development. For example, a planar polarized pattern of myosin II localization regulated by Rho1 signaling during Drosophila body axis elongation is thought to drive the cell behaviors that contribute to convergent extension. However, it is not well understood how specific aspects of a myosin localization pattern influence the multiple cell behaviors--including cell intercalation, cell shape changes, and apical cell area fluctuations--that simultaneously occur within a tissue during morphogenesis. Here, we use optogenetic activation (optoGEF) and deactivation (optoGAP) of Rho1 signaling to perturb the myosin pattern in the germband epithelium during Drosophila axis elongation and analyze the effects on contractile cell behaviors within the tissue. We find that uniform photoactivation of optoGEF or optoGAP is sufficient to rapidly override the endogenous myosin pattern, abolishing myosin planar polarity and reducing cell intercalation and convergent extension. However, these two perturbations have distinct effects on junctional and medial myosin localization, apical cell area fluctuations, and cell packings within the germband. Activation of Rho1 signaling in optoGEF embryos increases myosin accumulation in the medial-apical domain of germband cells, leading to increased amplitudes of apical cell area fluctuations. This enhanced contractility is translated into heterogeneous reductions in apical cell areas across the tissue, disrupting cellular packings within the germband. Conversely, inactivation of Rho1 signaling in optoGAP embryos decreases both medial and junctional myosin accumulation, leading to a dramatic reduction in cell area fluctuations. These results demonstrate that the level of Rho1 activity and the balance between junctional and medial myosin regulate apical cell area fluctuations and cellular packings in the germband, which have been proposed to influence the biophysics of cell rearrangements and tissue fluidity. STATEMENT OF SIGNIFICANCETissues are shaped by forces produced by dynamic patterns of actomyosin contractility. However, the mechanisms underlying these myosin patterns and their translation into cell behavior and tissue-level movements are not understood. Here, we show that optogenetic tools designed to control upstream regulators of myosin II can be used to rapidly manipulate myosin patterns and analyze the effects on cell behaviors during tissue morphogenesis. Combining optogenetics with live imaging in the developing fruit fly embryo, we show that acute perturbations to upstream myosin regulators are sufficient to rapidly perturb existing myosin patterns and alter cell movements and shapes during axis elongation, resulting in abnormalities in embryo shape. These results directly link myosin contractility patterns to cell behaviors that shape tissues, providing new insights into the mechanisms that generate functional tissues.

developmental biology↗