Biology subjects

Callegari, A.

Publications and source records attributed to Callegari, A..

5 recordsLinked to original sources

bioRxiv2021

Rapid generation of homozygous fluorescent knock-in human cells using CRISPR/Cas9 genome editing and validation by automated imaging and digital PCR screening

We have previously described a protocol for genome engineering of mammalian cultured cancer cells with CRISPR/Cas9 to generate homozygous knock-ins of fluorescent tags into endogenous genes 1. Here, we are updating this protocol to reflect major improvements in the workflow regarding efficiency and throughput. In brief, we have improved our method by combining high efficiency electroporation of optimized CRISPR/Cas9 reagents, screening of single cell derived clones by automated bright field and fluorescence imaging, rapidly assessing the number of tagged alleles and potential off-targets using digital PCR (dPCR) and automated data analysis. Compared to the original protocol 1, our current procedure (i) significantly increases the efficiency of tag integration, (ii) automates the identification of clones derived from single cells with correct subcellular localization of the tagged protein and (iii) provides a quantitative and high throughput assay to measure the number of on- and off-target integrations with dPCR. The increased efficiency of the new procedure reduces the number of clones that need to be analysed in- depth by more than ten-fold, and yields up to 20% of homozygous clones in polyploid cancer cell lines in a single genome engineering round. Overall, we were able to dramatically reduce the hands-on time from 30 days to 10 days during the overall [~]10 weeks procedure, allowing a single person to process up to 5 genes in parallel, assuming that validated reagents - e.g. PCR-primers, dPCR-assays, Western Blot antibodies - are available.

cell biology↗

bioRxiv2021

A quantitative map of nuclear pore assembly reveals two distinct mechanisms

Understanding how the nuclear pore complex (NPC) assembles is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during evolution of eukaryotes1-4. While at least two NPC assembly pathways exist, one during exit from mitosis and one during nuclear growth in interphase, we currently lack a quantitative map of the molecular assembly events. Here, we use fluorescence correlation spectroscopy (FCS) calibrated live imaging of endogenously fluorescently-tagged nucleoporins to map the changes in composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways employ strikingly different molecular mechanisms, inverting the order of addition of two large structural components, the central ring complex and nuclear filaments. The dynamic stoichiometry data underpinned integrative spatiotemporal modeling of the NPC assembly pathway, predicting the structures of postmitotic NPC assembly intermediates.

cell biology↗

bioRxiv2020

3D super-resolution fluorescence microscopy maps the variable molecular architecture of the Nuclear Pore Complex

Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of ~1000 proteins, termed nucleoporins, and has a mass of over 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of 3D super-resolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of over ten thousand individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ.

cell biology↗

bioRxiv2018

F-actin patches nucleated on chromosomes coordinate capture by microtubules in oocyte meiosis

Capture of each and every chromosome by spindle microtubules is essential to prevent chromosome loss and aneuploidy. In somatic cells, astral microtubules search and capture chromosomes forming lateral attachments to kinetochores. However, this mechanism alone is insufficient in large oocytes. We have previously shown that a contractile F-actin network is additionally required to collect chromosomes scattered in the 70-m starfish oocyte nucleus. How this F-actin-driven mechanism is coordinated with microtubule capture remained unknown. Here, we show that after nuclear envelope breakdown Arp2/3-nucleated F-actin patches form around chromosomes in a Ran-GTP-dependent manner, and we propose that these structures sterically block kinetochore-microtubule attachments. Once F-actin-driven chromosome transport is complete, coordinated disassembly of these F-actin patches allows synchronous capture by microtubules. Our observations indicate that this coordination is necessary, as early capture of chromosomes by microtubules would interfere with F-actin-driven transport leading to chromosome loss and formation of aneuploid eggs.

cell biology↗

bioRxiv2018

Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation

Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-{kappa}B (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an \"enhanceosome,\" nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.\n\nAuthor SummaryTo control transcription of a certain gene or a group of genes, both eukaryotes and prokaryotes express specialized proteins, transcription factors (TFs). During gene activation, TFs bind gene promotor sequences to recruit the transcriptional machinery including DNA polymerase II. TFs are often multi-subunit proteins containing a DNA-binding domain (DBD) as well as a protein-protein interaction interface. It was suggested that the duration of a TF-DNA binding event 1) depends on these two subunits and 2) dictates the outcome, i.e. the amount of mRNA produced from an activated gene. We set out to address these hypotheses using the transcriptional activator NF-{kappa}B (p65) as well as a number of mutants affecting different functional subunits. Using a combination of live-cell microscopy and RNA sequencing, we show that p65 DNA-binding time indeed correlates with the transcriptional output, but that this relationship depends on, and hence can be uncoupled by altering, the protein-protein interaction capacity. Our results suggest that, while p65 DNA binding times are dominated by the DBD, a transcriptional output can only be achieved with a functional protein-protein interaction subunit.

genetics↗