Computational solutions for omics data

High-throughput experimental technologies are generating increasingly massive and complex genomic data sets. The sheer enormity and heterogeneity of these data threaten to make the arising problems computationally infeasible. Fortunately, powerful algorithmic techniques lead to software that can answer important biomedical questions in practice. In this Review, we sample the algorithmic landscape, focusing on state-of-the-art techniques, the understanding of which will aid the bench biologist in analysing omics data. We spotlight specific examples that have facilitated and enriched analyses of sequence, transcriptomic and network data sets.National Institutes of Health (U.S.) (Grant GM081871

Polarity development by asymmetric protein-cluster distributions in response to cortical flows in C. elegans zygotes

Asymmetric cell-division in the one-cell embryo is a key step in embryonic development. The initially homogeneous zygote establishes an anterior-posterior axis within the cell, allowing for the unequal distribution of cell fate determinants and subsequent cell differentiation. Therefore, polarity development is a fundamental procedure that defines the information template from which all future cell processes derive their cues. Many molecular players in polarity formation in C. elegans have been identified, but the design principles that underpin their interactions and how this contributes to successful polarisation remain unclear. This thesis focuses on the role of the clustering species PAR-3 and how its integration in the governing biochemical network promotes robust polarisation. To correctly proceed to the two-cell stage, the foundational step of polarity formation must be responsive to the polarising cue and maintain established domains ready for downstream cell-cycle processes. We characterise global flows along the polarity axis and investigate coupling between the cortical flows and PAR-3 cluster sizes. We find there is no dynamic advantage for larger clusters and instead conclude all clusters flow with the same efficiency. Alternatively, we investigate whether enhancement of clusters is a response to mechanical forces within the cortex during the period of flow but find little direct evidence of this relationship. Rodriguez et al proposed kinase cycling between inactive (advective) and inactive (diffuse) state. We model two distinct reaction pathways through reaction-advection-diffusion simulation and assess their viability by implementation of Approximate Bayesian Computation. We find that direct binding through a flow-sensing, inactive state, followed by switching to a diffuse active state yields a network that is unviable and sensitive to perturbation. Sensitivity is alleviated when the advective species serves only to enhance independent binding of the active species. Therefore, we propose kinase cycling through this network motif as a mechanism towards enhanced robust polarisation.Open Acces

Toward a morphodynamic model of the cell: Signal processing for cell modeling

From a systems biology perspective, the cell is the principal element of information integration. Therefore, understanding the cell in its spatiotemporal context is the key to unraveling many of the still unknown mechanisms of life and disease. This article reviews image processing aspects relevant to the quantification of cell morphology and dynamics. We cover both acquisition (hardware) and analysis (software) related issues, in a multiscale fashion, from the detection of cellular components to the description of the entire cell in relation to its extracellular environment. We then describe ongoing efforts to integrate all this vast and diverse information along with data about the biomechanics of the cell to create a credible model of cell morphology and behavior.Carlos Ortiz-de-Solorzano and Arrate Muñoz-Barrutia were supported by the Spanish Ministry of Economy and Competitiveness grants with reference DPI2012-38090-C03-02 and TEC2013-48552-C02, respectively. Michal Kozubek was supported by the Czech Science Foundation (302/12/G157)

Lab-on-a-chip technologies for manipulation and imaging of C. elegans worms and embryos

The nematode Caenorhabditis elegans is an attractive model organism, owing notably to its short life cycle, genetic tractability, and optical transparency facilitating microscopic observation. This thesis deals with the realization of technological tools for the manipulation of worms and for studying thereby biologically relevant questions. The novel microfluidic devices that were developed are: #1 A microfluidic approach for size-dependent sorting of C. elegans nematodes on-chip. We take advantage of the external pressure-deformable profile of polydimethylsiloxane (PDMS) transfer channels that connect two on-chip worm chambers. The pressure-controlled effective cross-section of these channels creates adjustable filter structures that can be easily tuned for a specific worm sorting experiment, without changing the design parameters of the device itself. Considering that our sorting device is merely based on geometrical parameters and operated by simple fluidic and pressure control, we believe that it has strong potential for further use in advanced, automated, microfluidic C. elegans-based assay platforms. #2 A microfluidic device for studying signaling via diffusive secreted compounds between two specific C. elegans populations over prolonged durations. In particular, we designed a microfluidic assay to investigate the biological process of male-induced demise, i.e. lifespan shortening, in C. elegans hermaphrodites in the presence of a physically separated male population. For this purpose, male and hermaphrodite worm populations were confined in adjacent microchambers on the chip, whereas molecules secreted by males could be exchanged between both populations by periodically activating controlled fluidic transfer of ÎŒl-volume aliquots of male-conditioned medium. For male-conditioned hermaphrodites, we observe a reduction in mean lifespan of 4 days compared to non-conditioned on-chip culture. #3 Development of two reversible C. elegans immobilization methods for imaging applications. The first immobilization method takes advantage of a biocompatible and temperature-responsive hydrogel-microbead matrix. Our gel-based immobilization technique does not require a specific chip design and enables fast and reversible immobilization, thereby allowing successive imaging of the same single worm or of small worm populations at all development stages for several days. The second immobilization method takes advantage of the elastic properties of PDMS. We present two distinct microdevices, namely a micropillar array and a serpentine microchannel, for on-chip feeding and high-resolution imaging studies, respectively. Both devices consist of size-tunable PDMS structures that allow the same chips to be used for immobilization of worms at all development stages. Our microfluidic approach provides appropriate physiological conditions for long-term studies and enables worm recovery after the experiment. #4 A fully integrated microfluidic approach for the exploration of C. elegans early embryogenesis including the possibility of testing small-molecule inhibitors with increased throughput and versatility. Here, up to 100 embryos can be immobilized in parallel for simultaneous high-resolution time-lapse imaging of embryonic development from the 1-cell stage to hatching. We demonstrate time-controlled and reversible drug delivery to on-chip immobilized embryos, which is of relevance for biochemical and pharmacological assays

The functional network in predictive biology : predicting phenotype from genotype and predicting human disease from fungal phenotype

textThe ability to predict is one of the hallmarks of successful theories. Historically, the predictive power of biology has lagged behind disciplines like physics because the biological world is complex, challenging to quantify, and full of exceptions. However, in recent years the amount of available data has expanded exponentially and biological predictions based on this data become a possibility. The functional gene network is a quantitative way to integrate this data and a useful framework for making biological predictions. This study demonstrates that functional networks capture real biological insight and uses the network to predict both subcellular protein localization and the phenotypic outcome of gene knockouts. Furthermore, I use the functional network to evaluate genetic modules shared between diverse organisms that lead to orthologous phenotypes, many that are non-obvious. I show that the successful predictions of the functional network have broad applicability and implications that range from the design of large-scale biological experiments to the discovery of genes with potential roles in human disease.Institute for Cellular and Molecular Biolog

Population distribution analyses reveal a hierarchy of molecular players underlying parallel endocytic pathways.

Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis

Genetic screens in C. elegans for new modulators of protein homeostasis, with relevance for conformational diseases

Tese de doutoramento, Bioquímica (Genética Molecular), Universidade de Lisboa, Faculdade de Ciências, 2012Protein folding is an essential cellular process, required for proper molecular and cellular function. The cell has evolved as a sophisticated machinery that ensures the quality and stability of the proteome. The network of cellular processes that coordinates protein synthesis, folding, trafficking and clearance, and determines the fate of proteins that do not acquire a native conformation, is responsible for maintaining protein homeostasis (proteostasis) and is referred to as “the proteostasis network” (PN). The key components of the quality control system are molecular chaperones that ensure proper folding under physiological and stress conditions. To restore homeostasis, the PN also relies on stress sensors and inducible pathways, such as the heat shock response (HSR), the unfolded protein response (UPR) and the oxidative stress response. How a protein folds and acquires its native conformation is a matter of high medical relevance since a large number of human diseases are associated with protein misfolding. These conditions are broadly classified as conformational disorders, and they are caused either by genetic mutations that cause protein misfolding and premature degradation (e.g. Cystic Fibrosis and Gaucher’s disease); or by the accumulation of misfolded, aggregated and/or fibrillar protein inclusions that are toxic to the cell. In particular, the phenomenon of protein aggregation is a hallmark of a large number of neurodegenerative diseases (e.g. Alzheimer’s, Parkinson’s and Huntington's diseases and several ataxias), muscular dystrophies, metabolic disorders and certain types of cancer. Considerable efforts have been directed at dissecting of the mechanisms of protein aggregation and toxicity, but the full extent of events leading to cell dysfunction is still unclear (Chapter I). The unifying aspect of conformational disorders is, however, the inability of the PN to respond efficiently to misfolded and aggregation-prone proteins so as to prevent cellular toxicity. Therefore, it is urgent and relevant to multiple diseases to identify genetic modifiers that enhance proteostasis function and consequently prevent protein aggregation and toxicity. Research has benefited from powerful model systems that recapitulate important aspects of the human disease. In particular, the nematode Caenorhabditis elegans (C. elegans) is a tractable genetic model organism that combines sufficient complexity so as to allow research on both cellular and organismal (including behavioral) phenotypes, with simplicity that facilitates rapid, high‐throughput testing of hypotheses (Chapter I). Genetic screens performed to date have identified the network’s protective components which, when knocked down or deleted, lead to enhanced aggregation and/or toxicity. These include molecular chaperones, proteasome subunits, components of the autophagy machinery, and the stress-induced transcriptional regulators FOXO/DAF-16 and HSF-1. The work described in this thesis is novel as it focuses on the opposite side of the PN, i.e., the pathways that when down-regulated lead to enhanced folding capacity. We established a screening strategy in C. elegans using RNA interference (RNAi) to identify genetic modifiers that suppress protein aggregation and toxicity of multiple disease-related proteins (Chapter II). Our goal was to identify genes that, when downregulated, enhanced the functional properties of the proteostasis network and restored the folding environment. We thus identified 63 genetic modifiers that suppressed both polyglutamine (polyQ) and mutant superoxide dismutase I (SOD1) aggregation, of which only 23 also suppressed the toxicity phenotype associated with aggregation. This was an important finding as it demonstrated that aggregation and toxicity can be genetically uncoupled. From the initial hits, 9 modifiers systematically reduced the misfolding of endogenous metastable mutant proteins, suggesting a general improvement of the folding environment. We postulated that this effect could be a consequence of activation of the heat shock stress response and chaperone expression by the modifiers. Although, we found that 5 improved folding in a HSF-1/chaperone dependent manner, the remaining modifiers improved folding by altering metabolism and RNA processing functions. Overall, this study introduced new genetic modifiers that promote alternate cellular folding environments broadly protective against misfolding events. We then characterized further the genetic modifier gei-11, a negative regulator of the L-type acetylcholine receptor (AChR) at the neuromuscular junction, to determine the mechanism of proteostasis enhancement (Chapter III). Downregulation of gei-11 increased cholinergic signaling and calcium flux into the cytoplasm of muscle cells, via activation of the voltage-gated calcium channel, EGL-19, and the sarcoplasmic reticulum ryanodine receptor, UNC-68. This resulted in selective activation of HSF-1 and upregulation of cytosolic chaperones that restored the post-synaptic folding environment. Earlier work had identified a loss-of-function deletion mutation in unc-30 that regulates GABA expression in C. elegans neurons, and resulting in enhanced polyQ aggregation in post-synaptic muscle cells (Garcia et al. 2007). Notably, enhanced aggregation occurs when GABAergic signaling is completely inhibited, resulting in maximum cholinergic overstimulation of muscle cells, whereas suppression of aggregation results from a moderate increase in cholinergic signaling. The effects of increased AChR expression are not the same as complete inhibition of GABA signaling, in part because the signaling response (and degree of stimulation) occurs at an intermediate level through a titrated response and waves of Ca2+ release. Therefore, the effect on post-synaptic protein aggregation is a consequence of the degree of imbalance generated between ACh and GABA, with an apparent range for folding improvement by cholinergic signaling. We propose that altogether these studies underscore the importance of the balance between cholinergic and GABAergic signaling as a mechanism for non-autonomous neuronal regulation of proteostasis in postsynaptic cells, and provide compelling evidence that will lead to a better understanding of the control of stress responses through tissue signaling events, which is very relevant for a number of neuromuscular disorders. We have also initiated the characterization of the hit gene let-607 (Appendix II). This gene is predicted to encode the C. elegans ortholog of CREBh, an ER regulated transmembrane protein (RIP) bZIP transcription factor that maintains sterol homeostasis in the liver and mediates UPR. Downregulation of let-607 in C. elegans led to an improvement of proteostasis function through activation of the HSR, upregulation of molecular chaperones and consequent suppression of protein misfolding, in an HSF-1- and XBP-1-dependent manner. UPR induction was found to be epistatic and required for HSR activation by let-607 RNAi. This is not observed for other UPR inducers, revealing specificity of “crosstalk” between the two stress responses through let-607. Currently, we are further characterizing the role of let-607 on UPR and the mechanism involved in UPR-mediated activation of the cytosolic HSR. The studies presented in this thesis emphasize the value of genetic screens and model organisms for the identification of genes and pathways that maintain protein homeostasis and are compromised in disease. Our screening strategy and triage hypotheses revealed novel genes/pathways that can be modulated to improve the PN capacity and help resolving the issue of protein aggregation-toxicity. Even greater value is offered by complementation of these genetic studies with small molecule screens to ultimately identify the suitable targets for therapeutics. This is highlighted in the work on “Chaperone Therapeutics: Small Molecule Proteostasis Regulators of the Heat Shock Response for Protein Conformational Diseases” (Appendix III). In this work we describe the results of a ~900,000 small molecule screen that identified small molecule proteostasis regulator compounds (PRs) that induce HSF-1- dependent chaperone expression and restore protein folding in multiple conformational disease models. The enhancement of proteome stability by the PRs is mediated by HSF-1, DAF-16/FOXO, SKN-1/Nrf2 and the chaperone machinery, through mechanisms that are distinct from current known small molecule activators of the HSR. Together, genetic and chemical modulation of the PN reveal new candidates and new mechanisms to be targeted by PRs, establishing promising therapeutic approaches for a variety of protein conformational diseases.O folding proteico, processo pelo qual proteínas adquirem a sua conformação correcta, é essencial para o bom funcionamento molecular e celular. Assim, as células possuem um sofisticado mecanismo de controlo de qualidade do proteoma (i.e., a totalidade das proteínas da célula num dado instante). O conjunto de vias celulares que coordena a síntese, o folding, o tráfego e a degradação de proteínas, e que determina o destino de proteínas que não adquirem a conformação correcta, é responsável por promover e manter a homeostase proteica celular ("proteostasis network" ou PN). Os componentes principais deste sistema de controlo de qualidade são os chaperones moleculares, responsáveis pelo folding de proteínas quer em condições fisiológicas quer sob stress. A PN utiliza sensores e vias induzidas por stress para restabelecer a homeostase, como é o caso da heat shock response (HSR), da unfolded protein response (UPR) e a resposta ao stress oxidativo. O processo de folding, e o modo como cada proteína adquire a sua conformação nativa funcional tem uma enorme relevância clínica uma vez que existe um elevado número de doenças provocadas por misfolding proteico. Estas patologias são genericamente designadas por ‘doenças de conformação proteica’, e podem ser causadas por mutações que impedem uma dada proteína de adquirir a conformação correcta, o que leva à sua degradação prematura (ex: Fibrose Quística e doença de Gaucher); ou então podem ser a consequência da acumulação e agregação de proteínas na forma de inclusões ou fibras amilóides que são altamente tóxicas para a célula. O fenómeno de agregação proteica é característico de doenças neuro-degenerativas (ex: doenças de Alzheimer, Parkinson e Huntington e várias ataxias), distrofias musculares, doenças metabólicas e certos tipos de cancro. Têm sido desenvolvidos esforços consideráveis na investigação dos mecanismos responsáveis pela agregação, toxicidade e sintomas clínicos destas doenças, no entanto ainda existem muitas questões em aberto (Capítulo I). O aspecto comum entre as doenças de conformação proteica é, no entanto, a incapacidade das células ou da PN de resposta eficiente face à presença de proteínas com tendência para agregar, de modo a evitar a respectiva toxicidade. Assim, uma alternativa para fins terapêuticos à identificação da causa de toxicidade em cada doença em particular, poderá ser a identificação de moduladores genéticos que alteram e principalmente que melhoram a capacidade funcional da PN, consequentemente prevenindo agregação e toxicidade. Este tipo de trabalho tem tido imenso sucesso através da utilização de organismos modelo que apresentam fenótipos relevantes para estas patologias. Particularmente, o nemátode Caenorhabditis elegans (C. elegans) oferece inúmeras vantagens em termos de facilidade de manipulação genética, estudo de fenótipos celulares e organismais (incluindo comportamentais), e possibilidade de ser usado em high-throughput screens genéticos (Capítulo I). Os screens genéticos executados até a data identificaram já os principais componentes da PN com função protectora os quais, aquando se reduz a sua expressão, levam ao aumento de misfolding e agregação proteícos e/ou toxicidade. Tais componentes da PN incluem chaperones, componentes das vias de degradação, como o proteasoma e autofagia, e os factores de transcrição FOXO/DAF-16 e HSF-1 que participam na resposta a stress celular. O trabalho descrito nesta tese é inovador pois foca numa outra vertente da PN, ou seja, os componentes cuja redução da expressão leva a uma melhoria do folding global. Assim, foi estabelecida uma estratégia de screening em C. elegans, utilizando RNA de interferência (RNAi) para identificar moduladores genéticos que reduzem a formação de agregados e a toxicidade de múltiplas proteínas (Capítulo II). O objectivo deste trabalho foi identificar novos factores que, quando negativamente regulados por RNAi, melhoram a função da PN e consequentemente aumentam o folding. Foram inicialmente identificados 63 moduladores/RNAi que reduziram o fenótipo de agregação no modelo de expressão de poliglutaminas (polyQ) e da superóxido dismutase 1 mutante (SOD1). Destes, apenas 23 moduladores genéticos reduziram o fenótipo de toxicidade. Este resultado demonstra que os processos de agregação e toxicidade não têm de estar necessariamente acoplados. Por último, pusemos em evidência que 9 destes moduladores corrigiram dum modo consistente o folding de várias proteínas mutantes endógenas, extremamente susceptíveis a misfolding, sugerindo que os genes identificados são moduladores gerais do folding proteíco. Propusémos entāo que este efeito seja uma consequência da expressão de chaperones e outros componentes do controlo de qualidade celular que promovem folding. De facto, o efeito de 5 destes genes parece ser dependente do factor de transcrição HSF-1 e do aumento da expressão de chaperones. Os restantes parecem afectar o folding através de alterações metabólicas e de processamento de RNA. Em conclusão, este trabalho identificou novos moduladores de homeostase proteica, os quais actuam de forma a promover um novo “ambiente celular” que propicia uma maior capacidade de folding. De seguida, caracterizou-se o mecanismo pelo qual o modulador genético gei-11 melhora a capacidade de folding (Capítulo III). Mostrámos que a redução da expressão do gene gei-11, descrito como sendo um regulador negativo do receptor de acetilcolina tipo-L (L-AChR) na junção neuro-muscular de C. elegans, aumenta o sinal colinérgico nas células musculares, activando os canais de cálcio na membrana (EGL-19) e o influxo do cálcio para o citoplasma através do retículo sarcoplásmico. Subsequentemente, verificou-se que ocorria a activação da HSF-1 e a expressão de chaperones que, por sua vez, assistem o folding de proteínas e minimizam agregação. Por ouro lado, tinha já sido descrito que a deleção do gene unc-30, que regula a via de síntese e secreção de GABA em células neuronais de C. elegans, intensifica a agregação de poliQ nas células musculares (Apêndice II). De notar, é o facto do aumento da agregação ocorrer apenas quando o sinal GABAergico é completamente eliminado, o que resulta em estimulação colinérgica máxima das células musculares. Por outro lado, a redução no número de agregados resulta dum “aumento intermédio” de actividade colinérgica. O aumento de expressão de AChR através do gei-11/RNAi não é assim equivalente à total inibição do sinal GABAérgico, uma vez que no primeiro caso o efeito é de nível intermédio e com oscilação nos níveis de cálcio. Assim, a alteração dos níveis de agregação e uma consequência do grau de desequilíbrio entre ACh e GABA, com um aparente limite para efeito positivo no folding proporcionado por ACh. Deste modo, s duas componentes deste trabalho revelam que o equilíbrio entre o input neuronal GABAérgico e colinérgico é essencial para regulação da proteostase no músculo, e aparentemente revelam um limiar para a melhoria do folding por parte da sinalização colinérgica. Estes resultados também revelam assim duma forma clara as vias de comunicação entre o sistema nervoso e muscular, que se reveste de extrema relevância para um conjunto de doenças neuromusculares. No Apêndice III são também brevemente descritos os resultados da caracterização do modulador genético let-607. Prevê-se que este gene codifique o ortólogo do gene humano CREBh, um factor de transcrição associado à membrana do retículo endoplasmático (RE) e responsável pela regulação dos níveis de esteróis no fígado, bem como pela regulação da UPR. Observou-se que a redução da expressão de let-607 conduz à activação da UPR e da HSR, à expressão de chaperones dependentes de HSF-1 e XBP-1, e consequentemente uma melhoria do folding proteico. De notar que a UPR ocorre de forma epistatica relativamente à HSR, sob a acção do RNAi contra o gene let-607. Os resultados sugerem uma especificidade de comunicação (“crosstalk”) entre as duas vias de stress UPR e HSR, que é regulada pelos níveis de let-607. Presentemente, a função do let-607 na UPR e o mecanismo de activação da HSR pela UPR estão a ser caracterizados. O trabalho apresentado nesta tese sublinha o valor dos screens genéticos em organismos modelo para a identificação de novos factores e vias que contribuem para a homeostase celular. A estratégia de screening e o método de triagem aqui aplicados revelaram componentes específicos da PN que podem ser modificados com o objectivo de reduzir o fenótipo de agregação e toxicidade proteíca. Estes estudos genéticos podem ser complementados por screens de pequenas moléculas (“small molecules”) para identificação de compostos que sirvam de base para o desenvolvimento de novas terapêuticas. Este é o foco do trabalho publicado no artigo “Chaperone Therapeutics: Small Molecule Proteostasis Regulators of the Heat Shock Response for Protein Conformational Diseases” (Appendix IV). Nele é descrito o resultado dum screen de ~900.000 moléculas, a partir do qual foram identificados novos reguladores de proteostase (PRs) que activam a HSF-1 e a expressão de chaperones. A exposição de modelos celulares e animais de doenças de conformação proteica a estes PRs reduziu o misfolding de múltiplas proteínas e as respectivas consequências fenotípicas. Verificou-se que o melhoramento da capacidade de folding é regulado por HSF-1, DAF-16/FOXO, SKN-1/Nrf-2 e chaperones. No seu conjunto, este estudo revelou novos moduladores genéticos e químicos das vias de proteostase, os quais apresentam potencial terapêutico (por exemplo, os PRs) para várias doenças causadas por proteínas com conformações prejudiciais à célula/organismo

De novo identification of universal cell mechanics regulators

Mechanical proprieties determine many cellular functions, such as cell fate specification, migration, or circulation through vasculature. Identifying factors governing cell mechanical phenotype is therefore a subject of great interest. Here we present a mechanomics approach for establishing links between mechanical phenotype changes and the genes involved in driving them. We employ a machine learning-based discriminative network analysis method termed PC-corr to associate cell mechanical states, measured by real-time deformability cytometry (RT-DC), with large-scale transcriptome datasets ranging from stem cell development to cancer progression, and originating from different murine and human tissues. By intersecting the discriminative networks inferred from two selected datasets, we identify a conserved module of five genes with putative roles in the regulation of cell mechanics. We validate the power of the individual genes to discriminate between soft and stiff cell states in silico, and demonstrate experimentally that the top scoring gene, CAV1, changes the mechanical phenotype of cells when silenced or overexpressed. The data-driven approach presented here has the power of de novo identification of genes involved in cell mechanics regulation and paves the way towards engineering cell mechanical properties on demand to explore their impact on physiological and pathological cell functions