Analysis of the Architecture of the Nuclear Pore Complex by 3D super-resolution fluorescence microscopy

Nuclear pore complexes (NPCs), embedded in the two nuclear membranes, are the unique gateways that mediate all the traffic between the nucleus and the cytoplasm. In higher eukaryotes, each NPC is composed of multiple copies of approximately 30 different proteins termed nucleoporins and has a mass of over 100 MDa. In recent years, substantial effort has been devoted to the structural and functional characterization of this essential molecular machine in eukaryotic cells. Even though a pseudo-atomic model of the scaffold of the NPC has been produced, many details of the structure still remain elusive due to the enormous size, complexity and conformational dynamics of the NPC. In addition, we have little structural understanding of how such a large machine is assembled and what dynamic structural changes underlie its functions. During my PhD work, I established a methodology that can determine the 3D architecture of the NPC by combining single-molecule localization microscopy in a 4Pi detection scheme with computational classification and 3D single particle averaging. This new approach is able to resolve the structure of the NPC with molecular specificity and nano-scale resolution in situ in human cells. I here present the reconstruction of a 3D molecular map that integrates both scaffold and flexible nucleoporins and that allows us to address dynamic components of the NPC that have been inaccessible for atomic resolution methods to date. My findings indicate that the peripheral regions of the NPC can assume very different conformational states and that even the overall scaffold structure of the NPC is more flexible than previously assumed. The methodology I have established opens the exciting possibility to address novel structural, functional and assembly aspects of this fundamental cellular machine and can be applied to interrogate the 3D architecture of other large protein complexes and organelles inside cells

Sperm chemotaxis is driven by the slope of the chemoattractant concentration field.

Spermatozoa of marine invertebrates are attracted to their conspecific female gamete by diffusive molecules, called chemoattractants, released from the egg investments in a process known as chemotaxis. The information from the egg chemoattractant concentration field is decoded into intracellular Ca2+ concentration ([Ca2+]i) changes that regulate the internal motors that shape the flagellum as it beats. By studying sea urchin species-specific differences in sperm chemoattractant-receptor characteristics we show that receptor density constrains the steepness of the chemoattractant concentration gradient detectable by spermatozoa. Through analyzing different chemoattractant gradient forms, we demonstrate for the first time that Strongylocentrotus purpuratus sperm are chemotactic and this response is consistent with frequency entrainment of two coupled physiological oscillators: i) the stimulus function and ii) the [Ca2+]i changes. We demonstrate that the slope of the chemoattractant gradients provides the coupling force between both oscillators, arising as a fundamental requirement for sperm chemotaxis

An epigenetic switch regulates the ontogeny of AXL-positive/EGFR-TKi-resistant cells by modulating miR-335 expression.

Despite current advancements in research and therapeutics, lung cancer remains the leading cause of cancer-related mortality worldwide. This is mainly due to the resistance that patients develop against chemotherapeutic agents over the course of treatment. In the context of non-small cell lung cancers (NSCLC) harboring EGFR-oncogenic mutations, augmented levels of AXL and GAS6 have been found to drive resistance to EGFR tyrosine kinase inhibitors such as Erlotinib and Osimertinib in certain tumors with mesenchymal-like features. By studying the ontogeny of AXL-positive cells, we have identified a novel non-genetic mechanism of drug resistance based on cell-state transition. We demonstrate that AXL-positive cells are already present as a subpopulation of cancer cells in Erlotinib-naïve tumors and tumor-derived cell lines and that the expression of AXL is regulated through a stochastic mechanism centered on the epigenetic regulation of miR-335. The existence of a cell-intrinsic program through which AXL-positive/Erlotinib-resistant cells emerge infers the need of treating tumors harboring EGFR-oncogenic mutations upfront with combinatorial treatments targeting both AXL-negative and AXL-positive cancer cells

Real-time 3D single-molecule localization using experimental point spread functions.

We present a real-time fitter for 3D single-molecule localization microscopy using experimental point spread functions (PSFs) that achieves minimal uncertainty in 3D on any microscope and is compatible with any PSF engineering approach. We used this method to image cellular structures and attained unprecedented image quality for astigmatic PSFs. The fitter compensates for most optical aberrations and makes accurate 3D super-resolution microscopy broadly accessible, even on standard microscopes without dedicated 3D optics.</p