Lilia Espada, Alexander Dakhovnik, Prerana S Chaudhari, Asya Martirosyan, Laura Miek, Tetiana Poliezhaieva, Yvonne Schaub, Ashish Nair, Nadia Doering, Norman Rahnis, Oliver Werz, Andreas Koeberle, Joanna Kirkpatrick, Alessandro Ori, Maria Ermolaeva

The diabetes drug metformin is to be clinically tested in aged humans to achieve health span extension, but little is known about responses of old non-diabetic individuals to this drug. By in vitro and in vivo tests we found that metformin shortens life span and limits cell survival when provided in late life, contrary to its positive early life effects. Mechanistically, metformin exacerbates aging-associated mitochondrial dysfunction towards respiratory failure, aggravated by the inability of old cells to upregulate glycolysis in response to metformin, leading to ATP exhaustion. The beneficial dietary restriction effect of metformin on lipid reserves is abrogated in old animals, contributing to metabolic failure, while ectopic stabilization of cellular ATP levels alleviates late life metformin toxicity in vitro and in vivo. The toxicity is also suspended in nematodes carrying diabetes-like insulin receptor insufficiency and showing prolonged resilience to metabolic stress induced by metformin. In sum, we uncovered an alarming metabolic decay triggered by metformin in late life which may limit its benefits for non-diabetic elderly patients. Novel regulators of life extension by metformin are also presented.

Jeronimo Rodriguez-Beltran, Vidar Sørum, Macarena Toll-Riera, Carmen de la Vega, Rafael Peña-Miller, Alvaro San Millan

Mobile genetic elements (MGEs), such as plasmids, promote bacterial evolution through horizontal gene transfer (HGT). However, the rules governing the repertoire of traits encoded on MGEs remain unclear. In this study, we uncovered the central role of genetic dominance shaping genetic cargo in MGEs, using antibiotic resistance as a model system. MGEs are typically present in more than one copy per host bacterium and, as a consequence, genetic dominance favors the fixation of dominant mutations over recessive ones. Moreover, genetic dominance also determines the phenotypic effects of horizontally acquired MGE-encoded genes, silencing recessive alleles if the recipient bacterium already carries a wild-type copy of the gene. The combination of these two effects governs the catalogue of genes encoded on MGEs, dictating bacterial evolution through HGT.

Meenu SINGLA-RASTOGI, Magali CHARVIN, Odon Thiebeauld, Alvaro L Perez-Quintero, Antinea RAVET, Antonio EMIDIO-FORTUNATO, Venugopal MENDU, Lionel NAVARRO

Plant small RNAs (sRNAs) and/or double-stranded RNAs (dsRNAs) trigger RNA interference (RNAi) in interacting eukaryotic pathogens or parasites. However, it is unknown whether this phenomenon could operate in bacterial phytopathogens, which lack a eukaryoticlike RNAi machinery. Here, we first show that Arabidopsis-encoded inverted repeat transgenes trigger silencing of Pseudomonas syringae heterologous reporter and endogenous virulence-associated genes during infection. Antibacterial Gene Silencing (AGS) of the latter was associated with a reduced pathogenesis, which was also observed upon application of corresponding plant-derived RNAs onto wild-type plants prior to infection. We additionally demonstrate that sRNAs directed against virulence factor transcripts were causal for silencing and pathogenesis reduction, while cognate long dsRNAs were inactive. Overall, this study provides the first evidence that plant sRNAs can directly reprogram gene expression in a phytopathogenic bacterium and may have wider implications in the understanding of how plants regulate transcriptome, community composition and genome evolution of associated bacteria.

Thomas Gassler, Michael Sauer, Brigitte Gasser, Diethard Mattanovich, Matthias G Steiger

The methylotrophic yeast Pichia pastoris is frequently used for heterologous protein production and it assimilates methanol efficiently via the xylulose-5-phosphate pathway. This pathway is entirely localized in the peroxisomes and has striking similarities to the Calvin-Benson-Bassham (CBB) cycle, which is used by a plethora of organisms like plants to assimilate CO2 and is likewise compartmentalized in chloroplasts. By metabolic engineering the methanol assimilation pathway of P. pastoris was re-wired to a CO2 fixation pathway resembling the CBB cycle. This new yeast strain efficiently assimilates CO2 into biomass and utilizes it as its sole carbon source, which changes the lifestyle from heterotrophic to autotrophic. In total eight genes, including genes encoding for RuBisCO and phosphoribulokinase, were integrated into the genome of P. pastoris , while three endogenous genes were deleted to block methanol assimilation. The enzymes necessary for the synthetic CBB cycle were targeted to the peroxisome. Methanol oxidation, which yields NADH, is employed for energy generation defining the lifestyle as chemoorganoautotrophic. This work demonstrates that the lifestyle of an organism can be changed from chemoorganoheterotrophic to chemoorganoautotrophic by metabolic engineering. The resulting strain can grow exponentially and perform multiple cell doublings on CO2 as sole carbon source with a μmax of 0.008 h-1.

Harrison Specht, Edward Emmott, Aleksandra A. Petelski, R. Gray Huffman, David H. Perlman, Marco Serra, Peter Kharchenko, Antonius Koller, Nikolai Slavov

The fate and physiology of individual cells are controlled by protein interactions. Yet, our ability to quantitatively analyze proteins in single cells has remained limited. To overcome this barrier, we developed SCoPE2. It lowers cost and hands-on time by introducing automated and miniaturized sample preparation while substantially increasing quantitative accuracy. These advances enabled us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiated into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantified over 2,700 proteins in 1,018 single monocytes and macrophages in ten days of instrument time, and the quantified proteins allowed us to discern single cells by cell type. Furthermore, the data uncovered a continuous gradient of proteome states for the macrophage-like cells, suggesting that macrophage heterogeneity may emerge even in the absence of polarizing cytokines. Parallel measurements of transcripts by 10x Genomics scRNA-seq suggest that SCoPE2 samples 20-fold more copies per gene, thus supporting quantification with improved count statistics. Joint analysis of the data indicated that most genes had similar responses at the protein and RNA levels, though the responses of hundreds of genes differed. Our methodology lays the foundation for automated and quantitative single-cell analysis of proteins by mass-spectrometry. ![Figure][1]</img> [1]: pending:yes

Pille Hallast, Anastasia Agdzhoyan, Oleg Balanovsky, Yali Xue, Chris Tyler-Smith

The genomes of humans outside Africa originated almost entirely from a single migration out ~50,000-60,000 years ago1,2, followed closely by mixture with Neanderthals contributing ~2% to all non-Africans3,4. However, the details of this initial migration remain poorly-understood because no ancient DNA analyses are available from this key time period, and present-day autosomal data are uninformative due to subsequent population movements/reshaping5. One locus, however, does retain extensive information from this early period: the Y-chromosome, where a detailed calibrated phylogeny has been constructed6. Three present-day Y lineages were carried by the initial migration: the rare haplogroup D, the moderately rare C, and the very common FT lineage which now dominates most non-African populations6,7. We show that phylogenetic analyses of haplogroup C, D and FT sequences, including very rare deep-rooting lineages, together with phylogeographic analyses of ancient and present-day non-African Y-chromosomes, all point to East/South-east Asia as the origin 50,000-55,000 years ago of all known non-African male lineages (apart from recent migrants). This implies that the initial Y lineages in populations between Africa and eastern Asia have been entirely replaced by lineages from the east, contrasting with the expectations of the serial-founder model8,9, and thus informing and constraining models of the initial expansion.

Mark H Plitt, Lisa M Giocomo

The hippocampus is a medial temporal lobe brain structure that contains circuitry and neural representations capable of supporting declarative memory. Hippocampal place cells fire in one or few restricted spatial locations in a given environment. Between environmental contexts, place cell firing fields remap (turning on/off or moving to a new spatial location), providing a unique population-wide neural code for context specificity. However, the manner by which features associated with a given context combine to drive place cell remapping remains a matter of debate. Here we show that remapping of neural representations in region CA1 of the hippocampus is strongly driven by prior beliefs about the frequency of certain contexts, and that remapping is equivalent to an optimal estimate of the identity of the current context under that prior. This prior-driven remapping is learned early in training and remains robust to changes in behavioral task-demands. Furthermore, a simple associative learning mechanism is sufficient to reproduce these results. Our findings demonstrate that place cell remapping is a generalization of representing an animal's location. Rather than simply representing location in physical space, the hippocampus represents an optimal estimate of location in a multi-dimensional stimulus space.

Sydney B Blattman, Wenyan Jiang, Panos Oikonomou, Saeed Tavazoie

Despite longstanding appreciation of gene expression heterogeneity in isogenic bacterial populations, affordable and scalable technologies for studying single bacterial cells have been limited. While single-cell RNA sequencing (scRNA-seq) has revolutionized studies of transcriptional heterogeneity in diverse eukaryotic systems, application of scRNA-seq to prokaryotic cells has been hindered by their low levels of mRNA, lack of mRNA polyadenylation, and thick cell walls. Here, we present Prokaryotic Expression-profiling by Tagging RNA In Situ and sequencing (PETRI-seq), a high-throughput prokaryotic scRNA-seq pipeline that overcomes these obstacles. PETRI-seq uses in situ combinatorial indexing to barcode transcripts from tens of thousands of cells in a single experiment. We have demonstrated that PETRI-seq effectively captures single cell transcriptomes of Gram-negative and Gram-positive bacteria with high purity and little bias. Although bacteria express only thousands of mRNAs per cell, captured mRNA levels were sufficient to distinguish between the transcriptional states of single cells within isogenic populations. In E. coli, we were able to identify single cells in either stationary or exponential phase and define consensus transcriptomes for these sub-populations. In wild type S. aureus, we detected a rare population of cells undergoing prophage induction. We anticipate that PETRI-seq will be widely useful for studying transcriptional heterogeneity in microbial communities.

Balint Csorgo, Lina M Leon, Ilea J Chau-Ly, Alejandro Vasquez-Rifo, Joel D Berry, Caroline Mahendra, Emily Crawford, Jennifer D. Lewis, Joseph Bondy-Denomy

CRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we utilized a Cas3-based system featuring a processive nuclease, expressed endogenously or heterologously, for genome engineering purposes. Using an optimized and minimal CRISPR-Cas3 system (Type I-C) programmed with a single crRNA, large deletions ranging from 7 - 424 kb were generated in Pseudomonas aeruginosa with high efficiency and speed. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were variable in the absence of a homology-directed repair (HDR) template, and successfully and efficiently specified when present. The minimal Cas3 system is also portable; large deletions were induced with high efficiency in Pseudomonas syringae and Escherichia coli using an all-in-one vector. Notably, Cas3 generated bi-directional deletions originating from the programmed cut site, which was exploited to iteratively reduce a P. aeruginosa genome by 837 kb (13.5%) using 10 distinct crRNAs. We also demonstrate the utility of endogenous Cas3 systems (Type I-C and I-F) and develop an anti-anti-CRISPR strategy to circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid strain manipulation for synthetic biological and metabolic engineering purposes, genome minimization, and the analysis of large regions of unknown function.

Valeria Venturini, Fabio Pezzano, Frederic Català Castro, Hanna-Maria Häkkinen, Senda Jiménez-Delgado, Mariona Colomer-Rosell, Mónica Marro Sánchez, Queralt Tolosa-Ramon, Sonia Paz-López, Miguel A. Valverde, Pablo Loza-Alvarez, Michael Krieg, Stefan Wieser, Verena Ruprecht

The physical microenvironment regulates cell behavior during tissue development and homeostasis. How single cells decode information about their geometrical shape under mechanical stress and physical space constraints within their local environment remains largely unknown. Here we show that the nucleus, the biggest cellular organelle, functions as a non-dissipative cellular shape deformation gauge that enables cells to continuously measure shape variations on the time scale of seconds. Inner nuclear membrane unfolding together with the relative spatial intracellular positioning of the nucleus provides physical information on the amplitude and type of cellular shape deformation. This adaptively activates a calcium-dependent mechano-transduction pathway, controlling the level of actomyosin contractility and migration plasticity. Our data support that the nucleus establishes a functional module for cellular proprioception that enables cells to sense shape variations for adapting cellular behaviour to their microenvironment.

Maria I Rojas, Giselle S Cavalcanti, Katelyn McNair, Sean Benler, Amanda T Alker, Ana G Cobian-Guemes, Melissa Giluso, Kyle Levi, Forest Rohwer, Sinem Beyhan, Robert Edwards, Nicholas J Shikuma

An imbalance of normal bacterial groups such as Bacteroidales within the human gut is correlated with diseases like obesity. A current grand challenge in the microbiome field is to identify factors produced by normal microbiome bacteria that cause these observed health and disease correlations. While identifying factors like a bacterial injection system could provide a missing explanation for why Bacteroidales correlates with host health, no such factor has been identified to date. The lack of knowledge about these factors is a significant barrier to improving therapies like fecal transplants that promote a healthy microbiome. Here we show that a previously ill-defined Contractile Injection System is carried in the gut microbiome of 99% of individuals from the United States and Europe. This type of Contractile Injection System, we name here Bacteroidales Injection System (BIS), is related to the contractile tails of bacteriophage (viruses of bacteria) and have been described to mediate interactions between bacteria and diverse eukaryotes like amoeba, insects and tubeworms. Our findings that BIS are ubiquitous within adult human microbiomes suggest that they shape host health by mediating interactions between Bacteroidales bacteria and the human host or its microbiome.

Marta Maltese, Jeffrey R March, Alexander G Bashaw, Nicolas X Tritsch

Dopamine (DA) is a critical modulator of brain circuits that control voluntary movements, but our understanding of its influence on the activity of target neurons in vivo remains limited. Here, we use two-photon Ca2+ imaging to simultaneously monitor the activity of direct and indirect-pathway spiny projection neurons (SPNs) in the striatum of behaving mice during acute and prolonged manipulations of DA signaling. We find that, contrary to prevailing models, DA does not modulate activity rates in either pathway strongly or differentially. Instead, DA exerts a prominent influence on the overall number of direct and indirect pathway SPNs recruited during behavior. Chronic loss of midbrain DA neurons in a model of Parkinson's disease selectively impacts direct pathway ensembles and profoundly alters how they respond to DA elevation. Our results indicate that DA regulates striatal output by dynamically reconfiguring its sparse ensemble code and provide novel insights into the pathophysiology of Parkinson's disease.

Camille Marchet, Christina Boucher, Simon J Puglisi, Paul Medvedev, Mikael Salson, Rayan Chikhi

High-throughput sequencing datasets are usually deposited in public repositories, e.g. the European Nucleotide Archive, to ensure reproducibility. As the amount of data has reached petabyte scale, repositories do not allow to perform online sequence searches; yet such a feature would be highly useful to investigators. Towards this goal, in the last few years several computational approaches have been introduced to index and query large collections of datasets. Here we propose an accessible survey of these approaches, which are generally based on representing datasets as sets of k-mers. We review their properties, introduce a classification, and present their general intuition. We summarize their performance and highlight their current strengths and limitations.

Emily L Casanova, Julia L. Sharp, Stephen M Edelson, Desmond P. Kelly, Estate M. Sokhadze, Manuel F. Casanova

Background: A growing body of literature suggests etiological overlap between Ehlers-Danlos syndrome (EDS)/hypermobility spectrum disorders (HSD) and some cases of autism, although this relationship is poorly delineated. In addition, immune, autonomic, and endocrine dysregulation are reported in both conditions and may be relevant to their respective etiologies. Aims: To study symptom overlap in these two comorbid spectrum conditions. Methods and Procedures: We surveyed 702 adults aged 25+ years on a variety of EDS/HSD-related health topics, comparing individuals with EDS/HSD, autism, and unaffected controls. Outcomes and Results: The autism group reported similar though less severe symptomology as the EDS/HSD group, especially in areas of immune/autonomic/endocrine dysregulation, connective tissue abnormalities (i.e., skin, bruising/bleeding), and chronic pain. EDS/HSD mothers with autistic children reported more immune symptoms than EDS/HSD mothers without, suggesting the maternal immune system could play a heritable role in these conditions (p = 0.0119). Conclusions and Implications: These data suggest that EDS/HSD and autism share aspects of immune/autonomic/endocrine dysregulation, pain, and some tissue fragility, which is typically more severe in the former. This overlap, as well as documented comorbidity, suggests some forms of autism may be hereditary connective tissue disorders (HCTD).

Daniel Richter, Romain Watteaux, Thomas Vannier, Jade Leconte, Paul Frémont, Gabriel Reygondeau, Nicolas Maillet, Nicolas Henry, Gaëtan Benoit, Antonio Fernàndez-Guerra, Samir Suweis, Romain Narci, Cédric Berney, Damien Eveillard, Frederick Gavory, Lionel Guidi, Karine Labadie, Eric Mahieu, Julie Poulain, Sarah Romac, Simon Roux, Céline Dimier, Stefanie Kandels, Marc Picheral, Sarah Searson, &lt;I&gt;Tara&lt;/I&gt; Oceans Coordinators, Stéphane Pesant, Jean-Marc Aury, Jennifer R Brum, Claire Lemaitre, Eric Pelletier, Peer Bork, Shinichi Sunagawa, Lee Karp-Boss, Chris Bowler, Matthew B Sullivan, Eric Karsenti, Mahendra Mariadassou, Ian Probert, Pierre Peterlongo, Patrick Wincker, Colomban de Vargas, Maurizio Ribera d'Alcalà, Daniele Iudicone, Olivier Jaillon

Biogeographical studies have traditionally focused on readily visible organisms, but recent technological advances are enabling analyses of the large-scale distribution of microscopic organisms, whose biogeographical patterns have long been debated1,2. The most prominent global biogeography of marine plankton was derived by Longhurst3 based on parameters principally associated with photosynthetic plankton. Localized studies of selected plankton taxa or specific organismal sizes1,4-7 have mapped community structure and begun to assess the roles of environment and ocean current transport in shaping these patterns2,8. Here we assess global plankton biogeography and its relation to the biological, chemical and physical context of the ocean (the 'seascape') by analyzing 24 terabases of metagenomic sequence data and 739 million metabarcodes from the Tara Oceans expedition in light of environmental data and simulated ocean current transport. In addition to significant local heterogeneity, viral, prokaryotic and eukaryotic plankton communities all display near steady-state, large-scale, size-dependent biogeographical patterns. Correlation analyses between plankton transport time and metagenomic or environmental dissimilarity reveal the existence of basin-scale biological and environmental continua emerging within the main current systems. Across oceans, there is a measurable, continuous change within communities and environmental factors up to an average of 1.5 years of travel time. Modulation of plankton communities during transport varies with organismal size, such that the distribution of smaller plankton best matches Longhurst biogeochemical provinces, whereas larger plankton group into larger provinces. Together these findings provide an integrated framework to interpret plankton community organization in its physico-chemical context, paving the way to a better understanding of oceanic ecosystem functioning in a changing global environment.

Martin Smith, Tansel Ersavas, James M. Ferguson, Huanle Liu, Morghan C Lucas, Oguzhan Begik, Lilly Bojarski, Kirston Barton, Eva Maria Novoa

Nanopore sequencing has enabled sequencing of native RNA molecules without conversion to cDNA, thus opening the gates to a new era for the unbiased study of RNA biology. However, a formal barcoding protocol for direct sequencing of native RNA molecules is currently lacking, limiting the efficient processing of multiple samples in the same flowcell. A major limitation for the development of barcoding protocols for direct RNA sequencing is the error rate introduced during the base-calling process, especially towards the 5' and 3' ends of reads, which complicates sequence-based barcode demultiplexing. Here, we propose a novel strategy to barcode and demultiplex direct RNA sequencing nanopore data, which does not rely on base-calling or additional library preparation steps. Specifically, custom DNA oligonucleotides are ligated to RNA transcripts during library preparation. Then, raw current signal corresponding to the DNA barcode is extracted and transformed into an array of pixels, which is used to determine the underlying barcode using a deep convolutional neural network classifier. Our method, DeePlexiCon, implements a 20-layer residual neural network model that can demultiplex 93% of the reads with 95.1% specificity, or 60% of reads with 99.9% specificity. The availability of an efficient and simple barcoding strategy for native RNA sequencing will enhance the use of direct RNA sequencing by making it more cost-effective to the entire community. Moreover, it will facilitate the applicability of direct RNA sequencing to samples where the RNA amounts are limited, such as patient-derived samples.

I. Khait, Ohad Lewin-Epstein, R. Sharon, K. Saban, R. Perelman, A. Boonman, Y. Yovel, L. Hadany

Stressed plants show altered phenotypes, including changes in color, smell, and shape. Yet, the possibility that plants emit airborne sounds when stressed – similarly to many animals – has not been investigated. Here we show, to our knowledge for the first time, that stressed plants emit airborne sounds that can be recorded remotely, both in acoustic chambers and in greenhouses. We recorded ∼65 dBSPL ultrasonic sounds 10 cm from tomato and tobacco plants, implying that these sounds could be detected by some organisms from up to several meters away. We developed machine learning models that were capable of distinguishing between plant sounds and general noises, and identifying the condition of the plants – dry, cut, or intact – based solely on the emitted sounds. Our results suggest that animals, humans, and possibly even other plants, could use sounds emitted by a plant to gain information about the plant’s condition. More investigation on plant bioacoustics in general and on sound emission in plants in particular may open new avenues for understanding plants and their interactions with the environment, and it may also have a significant impact on agriculture.

Laura Pritschet, Tyler Santander, Evan Layher, Caitlin M Taylor, Shuying Yu, Michael B. Miller, Scott T Grafton, Emily G Jacobs

The brain is an endocrine organ, sensitive to the rhythmic changes in sex hormone production that occurs in most mammalian species. In rodents and nonhuman primates, estrogen and progesterone's impact on the brain is evident across a range of spatiotemporal scales. Yet, the influence of sex hormones on the functional architecture of the human brain is largely unknown. In this dense-sampling, deep phenotyping study, we examine the extent to which endogenous fluctuations in sex hormones alter intrinsic brain networks at rest in a woman who underwent brain imaging and venipuncture for 30 consecutive days. Standardized regression analyses illustrate estrogen and progesterone's widespread influence on cortical dynamics. Time-lagged analyses examined the temporal directionality of these relationships and reveal estrogen's ability to drive connectivity across major functional brain networks, including the Default Mode and Dorsal Attention Networks, whose hubs are densely populated with estrogen receptors. These results reveal the rhythmic nature in which brain networks reorganize across the human menstrual cycle. Neuroimaging studies that densely sample the individual connectome have begun to transform our understanding of the brain's functional organization. As these results indicate, taking endocrine factors into account is critical for fully understanding the intrinsic dynamics of the human brain.

Wilma A. Bainbridge, Zoë Pounder, Alison F Eardley, Chris I. Baker

Congenital aphantasia is a recently identified experience defined by the inability to form voluntary visual imagery, with intact semantic memory and vision. Although understanding aphantasia promises insights into the nature of visual imagery, as a new focus of study, research is limited and has largely focused on small samples and subjective report. The current large-scale online study of aphantasics (N=63) and controls required participants to draw real-world scenes from memory, and copy them during a matched perceptual condition. Drawings were objectively quantified by 2,700 online scorers for object and spatial details. Aphantasics recalled significantly fewer object details than controls, and showed a reliance on verbal strategies. However, aphantasics showed equally high spatial accuracy as controls, and made significantly fewer memory errors, with no differences between groups in the perceptual condition. This object-specific memory impairment in aphantasics provides evidence for separate systems in memory that support object versus spatial details.

Alexis Lomakin, Cedric Cattin, Damien Cuvelier, Zahraa Alraies, Marc Molina, Guilherme Nader, Nishit Srivastava, Juan-Manuel Garcia Arcos, Irina Zhitnyak, Anvita Bhargava, Meghan K Driscoll, Erik S Welf, Reto Paul Fiolka, Ryan Petrie, Nicolas Manel, Ana-Maria Paul Lennon-Dumenil, Daniel Muller, Matthieu Piel

The microscopic environment inside a metazoan organism is highly crowded. Whether individual cells can tailor their behavior to the limited space remains unclear. Here, we found that cells measure the degree of spatial confinement using their largest and stiffest organelle, the nucleus. Cell confinement below a resting nucleus size deforms the nucleus, which expands and stretches its envelope. This activates signaling to the actomyosin cortex via nuclear envelope stretch-sensitive proteins, upregulating cell contractility. We established that the tailored contractile response constitutes a nuclear ruler-based signaling pathway involved in migratory cell behaviors. Cells rely on the nuclear ruler to modulate the motive force enabling their passage through restrictive pores in complex three-dimensional (3D) environments, a process relevant to cancer cell invasion, immune responses and embryonic development.