Biology subjects

Monterroso, B.

Publications and source records attributed to Monterroso, B..

7 recordsLinked to original sources

bioRxiv2025

Mirror-image inversion in commonly used compound microscopes

Optical microscopes are essential tools for magnifying and analyzing a wide range of samples and are expected to faithfully preserve the original object in all its dimensions. However, while examining cerebral structures with left-right asymmetry, we discovered that the image of the object can be unintentionally mirror-inverted, depending on the microscope setup. This inversion mostly goes unnoticed, leading to incorrectly oriented images, loss of image integrity, and potential misinterpretation. We detail the optical origins of this issue and offer a practical guide to support accurate sample imaging, while urging manufacturers to explicitly communicate how their systems affect image orientation.

developmental biology↗

bioRxiv2024

Bacterial cell cycle regulator MatP forms biomolecular condensates and directly interacts with cell division protein FtsZ

An increasing number of proteins involved in bacterial cell cycle events have been recently shown to undergo phase separation. The resulting biomolecular condensates play an important role in cell cycle protein function and may be involved in development of persister cells tolerant to antibiotics. Here we report that the E. coli chromosomal Ter macrodomain organizer MatP, a division site selection protein implicated in the coordination of chromosome segregation with cell division, forms biomolecular condensates in cytomimetic systems. These condensates are favored by crowding and preferentially localize at the membrane of microfluidics droplets, a behavior probably mediated by MatP-lipid binding. Condensates are negatively regulated and partially dislodged from the membrane by DNA sequences recognized by MatP (matS), which partition into them. Unexpectedly, MatP condensation is enhanced by FtsZ, a core component of the division machinery previously described to undergo phase separation. Our biophysical analyses uncover a direct interaction between the two proteins, disrupted by matS sequences. This binding might have implications for FtsZ ring positioning at mid-cell by the Ter linkage, which comprises MatP and two other proteins that bridge the canonical MatP/FtsZ interaction. FtsZ/MatP condensates interconvert with bundles in response to GTP addition, providing additional levels of regulation. Consistent with discrete foci reported in cells, MatP biomolecular condensates may facilitate MatPs role in chromosome organization and spatiotemporal regulation of cytokinesis and DNA segregation. Moreover, sequestration of MatP in these membraneless compartments, with or without FtsZ, could promote cell entry into dormant states that are able to survive antibiotic treatments.

biochemistry↗

bioRxiv2022

Tethering by Uso1 is dispensable: The Uso1 monomeric globular head domain interacts with SNAREs to maintain viability.

Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Paradoxically, genetic studies placed Uso1 acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S substitutions in the GHD permitting growth of otherwise inviable rab1-deficient Aspergillus nidulans. Remarkably, the double mutant suppresses the complete absence of RAB1. Full-length Uso1 and CTR{Delta} proteins are dimeric and the GHD lacking the CC/CTR is monomeric irrespective of whether they carry or not E6K/G540S. Microscopy showed recurrence of Uso1 on puncta (60 sec half-life) colocalizing with RAB1 and less so with early Golgi markers Sed5 and GeaA/Gea1/Gea2. Localization of Uso1 but not of Uso1E6K/G540S to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. By S-tag-coprecipitation we demonstrate that Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Bos1 and Bet1 bind the Uso1 GHD directly, but Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. AlphaFold2 predicts that G540S actually increases binding of GHD to the Bos1 Habc domain. In contrast, E6K seemingly increases membrane targeting of an N-terminal amphipathic -helix, explaining phenotypic additivity. Overexpression of E6K/G540S and wild-type GHD complemented uso1{Delta}. Thus, a GHD monomer provides the essential Uso1 functions, demonstrating that long-range tethering activity is dispensable. Therefore, when enhanced by E6K/G540S, Uso1 binding to Bos1/Bet1 required to regulate SNAREs bypasses both the contribution of RAB1 to Uso1 recruitment and the reported role of RAB1 in SNARE complex formation (Lupashin and Waters, 1997), suggesting that the latter is consequence of the former.

cell biology↗

bioRxiv2021

Long-term quality assessment and monitoring of light microscope performance through accessible and reliable protocols, tools and metrics

Reliable, reproducible and comparable results are what biology requires from microscopy. To achieve that level of confidence, monitoring the stability of the microscope performance over time with standardized quality testing routines is essential for mining quantitative data. Three levels of microscope quality control procedures should be considered: i) usage of accessible and affordable tools and samples, ii) execution of easy and fast, preferably automatized, acquisition protocols, iii) analysis of data in the most automated way possible with adequate metrics for long-term monitoring. In this paper, we test the acquisition protocols on the mainly used microscope techniques (wide-field, spinning disk and confocal microscopy) with simple quality control tools. Seven protocols specify metrics on measuring the lateral and axial resolution (Point-Spread Function) of the system, field flatness, chromatic aberrations and co-registration, illumination power monitoring and stability, stage drift and positioning repeatability and finally temporal and spatial noise sources of camera detectors. We designed an ImageJ/FiJi java plugin named MetroloJ_QC to incorporate the identified metrics and automatize the data processing for the analysis. After processing and comparing the data of microscopes from more than ten imaging facilities, we test the robustness of the metrics and the protocols by determining experimental limit values. Our results give a first extensive characterization of the quality control procedures of a light microscope, with an automated data processing and experimental limit values that can be used by core facility staff and researchers to monitor the microscope performance over time.

scientific communication and education↗

bioRxiv2020

Assembly of bacterial cell division protein FtsZ into dynamic biomolecular condensates

Biomolecular condensation through phase separation may be a novel mechanism to regulate bacterial processes, including cell division. Previous work revealed FtsZ, a protein essential for cytokinesis in most bacteria, and the E. coli division site selection factor SlmA form FtsZ*SlmA biomolecular condensates. The absence of condensates composed solely of FtsZ under the conditions used in that study suggested this mechanism was restricted to nucleoid occlusion or SlmA-containing bacteria. Here we report that FtsZ alone can demix into condensates in bulk and when encapsulated in synthetic cell-like systems. Condensate assembly depends on FtsZ being in the GDP-bound state and on crowding conditions that promote its oligomerization. FtsZ condensates are dynamic and gradually convert into FtsZ filaments upon GTP addition. Notably, FtsZ lacking its C-terminal disordered region, a structural element likely to favor biomolecular condensation, also forms condensates, albeit less efficiently. The inherent tendency of FtsZ to form condensates susceptible to modulation by physiological factors, including binding partners, suggests that such mechanisms may play a more general role in bacterial cell division than initially envisioned.

biochemistry↗

bioRxiv2018

The bacterial DNA binding protein MatP involved in linking the nucleoid terminal domain to the divisome at midcell interacts with lipid membranes

Division ring formation at midcell is controlled by various mechanisms in Escherichia coli, one of them being the linkage between the chromosomal Ter macrodomain and the Z-ring mediated by MatP, a DNA binding protein that organizes this macrodomain and contributes to the prevention of premature chromosome segregation. Here we show that, during cell division, just before splitting the daughter cells, MatP seems to localize close to the cytoplasmic membrane, suggesting that this protein might interact with lipids. To test this hypothesis, we investigated MatP interaction with lipids in vitro. We found that MatP, when encapsulated inside microdroplets generated by microfluidics and giant vesicles, accumulates at phospholipid bilayers and monolayers matching the lipid composition in the E. coli inner membrane. MatP binding to lipids was independently confirmed using lipid coated microbeads and bio-layer interferometry assays. Interaction of MatP with the lipid membranes also occurs in the presence of the DNA sequences specifically targeted by the protein but there is no evidence of ternary membrane/protein/DNA complexes. We propose that the interaction of MatP with lipids may modulate its spatiotemporal localization and its recognition of other ligands.\n\nIMPORTANCEThe division of an E. coli cell into two daughter cells with equal genomic information and similar size requires duplication and segregation of the chromosome and subsequent scission of the envelope by a protein ring, the Z-ring. MatP is a DNA binding protein that contributes both to the positioning of the Z-ring at midcell and the temporal control of nucleoid segregation. Our integrated in vivo and in vitro analysis provides evidence that MatP can interact with lipid membranes comprising the phospholipid mixture in the E. coli inner membrane, without concomitant recruitment of the short DNA sequences specifically targeted by MatP. This observation strongly suggests that the membrane may play a role in the regulation of the function and localization of MatP, which could be relevant for the coordination of the two fundamental processes in which this protein participates, nucleoid segregation and cell division.

biochemistry↗

bioRxiv2018

Bacterial division FtsZ forms liquid condensates with nucleoid-associated Z-ring inhibitor SlmA

Macromolecular condensation resulting from biologically regulated liquid-liquid phase transitions is emerging as a mechanism to organize the intracellular space in eukaryotic systems, with broad implications in cell physiology and pathology. Here we show that FtsZ, central element of the division ring in most bacteria, forms condensates when in complex with SlmA, the protein preventing septal ring assembly nearby the chromosome in E. coli. The formation of condensates is promoted by crowding and enhanced by sequence-specific binding of SlmA to DNA. These structures are dynamic and FtsZ within them remains active for GTP-triggered fiber formation. Their location is sensitive to compartmentalization and to the presence of a membrane boundary in microfluidics-based cell mimetic systems, likely affecting their reactivity. We propose that reversible condensation may play a role in the modulation of FtsZ assembly and/or location by SlmA and, hence, in the regulation of ring stability, constituting a singular example of a prokaryotic nucleoprotein complex exhibiting this kind of phase transition.

biochemistry↗