RESUMEN
Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution, yet how they form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. Using fluorescence correlation spectroscopy we find there are spatial differences in Bcd diffusivity along the AP axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. In the nucleus, we demonstrate that Bcd dynamics are impacted by binding to DNA. Addition of the Bcd homeodomain to eGFP::NLS qualitatively replicates the Bcd concentration profile, suggesting this domain regulates Bcd dynamics. Our results reveal how a long-range gradient can form while retaining a steep profile through much of its range.
Asunto(s)
Proteínas de Drosophila , Proteínas de Homeodominio , Animales , Tipificación del Cuerpo/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
T cells comprise functionally diverse subtypes. Although activated via a conserved scheme of antigen recognition by their T cell receptor, they elicit heterogeneous activation and effector responses. Such functional diversity has been appreciated in gene expression studies, functional assays, and disease models. Yet, our understanding of the principles underlying T cell subtype-specific activation and antigen recognition in the immunological synapse remains limited. This is primarily due to difficulties in primary T cell visualization at high spatiotemporal resolution and the adoption of tractable transformed T cell systems for cell biological experiments that may not correctly represent primary T cell constitutional diversity. Here, we discuss recent findings regarding the architectural and dynamic diversity of the immunological synapse and state-of-the-art methodologies that can be utilized to provide clues on how biological and biophysical differences in synaptic make-up could govern functional divergences in T cell subtypes.
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Linfocitos B/inmunología , Células Dendríticas/inmunología , Sinapsis Inmunológicas/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Humanos , Activación de Linfocitos , Receptor Cross-Talk , Transducción de SeñalRESUMEN
Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Células Dendríticas/inmunología , Estrés del Retículo Endoplásmico/inmunología , Activación de Linfocitos , Células T Asesinas Naturales/inmunología , Animales , Presentación de Antígeno , Antígenos CD1d/biosíntesis , Antígenos CD1d/inmunología , Autoantígenos/inmunología , Carcinoma Pulmonar de Lewis/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Citoesqueleto/ultraestructura , Endosomas/inmunología , Glicoesfingolípidos/inmunología , Glicoesfingolípidos/metabolismo , Humanos , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Lípidos/inmunología , Lisosomas/inmunología , Ratones , Ratones Endogámicos C57BL , Células THP-1 , Tapsigargina/farmacología , Respuesta de Proteína Desplegada/inmunología , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/fisiologíaRESUMEN
Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.
Asunto(s)
Citoesqueleto de Actina/fisiología , Endotelio Linfático/metabolismo , Endotelio Vascular/metabolismo , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células Cultivadas , Endotelio Linfático/citología , Endotelio Vascular/citología , Humanos , Receptores de Hialuranos/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
Cytoskeletal actin dynamics are crucial for the activation of T-cells. Immortalised Jurkat T-cells have been the model system of choice to examine and correlate the dynamics of the actin cytoskeleton and the immunological synapse leading to T-cell activation. However, it has remained unclear whether immortalised cellular systems, such as Jurkat T-cells can recapitulate the cytoskeletal behaviour of primary T-cells. Studies delineating the cytoskeletal behaviour of Jurkat T-cells in comparison to primary T-cells are lacking. Here, we employ live-cell super-resolution microscopy to investigate the cytoskeletal actin organisation and dynamics of living primary and immortalised Jurkat T-cells at the appropriate spatiotemporal resolution. Under comparable activation conditions, we found differences in the architectural organisation and dynamics of Jurkat and primary mouse and human T-cells. Although the three main actin network architectures in Jurkat T-cells were reminiscent of primary T-cells, there were differences in the organisation and molecular mechanisms underlying these networks. Our results highlight mechanistic distinctions in the T-cell model system most utilised to study cytoskeletal actin dynamics.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sinapsis Inmunológicas/metabolismo , Linfocitos T/citología , Animales , Reordenamiento Génico de Linfocito T , Humanos , Células Jurkat , Activación de Linfocitos , Ratones , Modelos Biológicos , Receptores de Antígenos de Linfocitos T/genética , Transducción de SeñalRESUMEN
Quantifying cell generated mechanical forces is key to furthering our understanding of mechanobiology. Traction force microscopy (TFM) is one of the most broadly applied force probing technologies, but its sensitivity is strictly dependent on the spatio-temporal resolution of the underlying imaging system. In previous works, it was demonstrated that increased sampling densities of cell derived forces permitted by super-resolution fluorescence imaging enhanced the sensitivity of the TFM method. However, these recent advances to TFM based on super-resolution techniques were limited to slow acquisition speeds and high illumination powers. Here, we present three novel TFM approaches that, in combination with total internal reflection, structured illumination microscopy and astigmatism, improve the spatial and temporal performance in either two-dimensional or three-dimensional mechanical force quantification, while maintaining low illumination powers. These three techniques can be straightforwardly implemented on a single optical set-up offering a powerful platform to provide new insights into the physiological force generation in a wide range of biological studies. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.
Asunto(s)
Microscopía de Fuerza Atómica/métodos , Microscopía Fluorescente/métodos , Animales , Fenómenos Biofísicos , Adhesión Celular/fisiología , Fenómenos Fisiológicos Celulares , Simulación por Computador , Humanos , Imagenología Tridimensional , Luz , Fenómenos Mecánicos , Microscopía de Fuerza Atómica/instrumentación , Microscopía de Fuerza Atómica/estadística & datos numéricos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/estadística & datos numéricos , Análisis Espacio-TemporalRESUMEN
Quantification of mechanical forces is a major challenge across biomedical sciences. Yet such measurements are essential to understanding the role of biomechanics in cell regulation and function. Traction force microscopy remains the most broadly applied force probing technology but typically restricts itself to single-plane two-dimensional quantifications with limited spatiotemporal resolution. Here, we introduce an enhanced force measurement technique combining 3D super-resolution fluorescence structural illumination microscopy and traction force microscopy (3D-SIM-TFM) offering increased spatiotemporal resolution, opening-up unprecedented insights into physiological three-dimensional force production in living cells.
Asunto(s)
Simulación por Computador , Microscopía de Fuerza Atómica , TracciónRESUMEN
Quantifying the adaptive mechanical behavior of living cells is essential for the understanding of their inner working and function. Yet, despite the establishment of quantitative methodologies correlating independent measurements of cell mechanics and its underlying molecular kinetics, explicit evidence and knowledge of the sensitivity of the feedback mechanisms of cells controlling their adaptive mechanics behavior remains elusive. Here, a combination of atomic force microscopy and fluorescence recovery after photobleaching is introduced offering simultaneous quantification and direct correlation of molecule kinetics and mechanics in living cells. Systematic application of this optomechanical atomic force microscopy-fluorescence recovery after photobleaching platform reveals changes in the actin turnover and filament lengths of ventral actin stress fibers in response to constant mechanical force at the apical actin cortex with a dynamic range from 0.1 to 10 nN, highlighting a direct relationship of active mechanosensation and adaptation of the cellular actin cytoskeleton. Simultaneous quantification of the relationship between molecule kinetics and cell mechanics may thus open-up unprecedented insights into adaptive mechanobiological mechanisms of cells.
Asunto(s)
Células/metabolismo , Actinas/metabolismo , Fenómenos Biomecánicos , Calibración , Recuperación de Fluorescencia tras Fotoblanqueo , Células HEK293 , Células HeLa , Humanos , Microscopía de Fuerza Atómica , Fibras de Estrés/metabolismoRESUMEN
Developments in microscopy have been instrumental to progress in the life sciences, and many new techniques have been introduced and led to new discoveries throughout the last century. A wide and diverse range of methodologies is now available, including electron microscopy, atomic force microscopy, magnetic resonance imaging, small-angle x-ray scattering and multiple super-resolution fluorescence techniques, and each of these methods provides valuable read-outs to meet the demands set by the samples under study. Yet, the investigation of cell development requires a multi-parametric approach to address both the structure and spatio-temporal organization of organelles, and also the transduction of chemical signals and forces involved in cell-cell interactions. Although the microscopy technologies for observing each of these characteristics are well developed, none of them can offer read-out of all characteristics simultaneously, which limits the information content of a measurement. For example, while electron microscopy is able to disclose the structural layout of cells and the macromolecular arrangement of proteins, it cannot directly follow dynamics in living cells. The latter can be achieved with fluorescence microscopy which, however, requires labelling and lacks spatial resolution. A remedy is to combine and correlate different readouts from the same specimen, which opens new avenues to understand structure-function relations in biomedical research. At the same time, such correlative approaches pose new challenges concerning sample preparation, instrument stability, region of interest retrieval, and data analysis. Because the field of correlative microscopy is relatively young, the capabilities of the various approaches have yet to be fully explored, and uncertainties remain when considering the best choice of strategy and workflow for the correlative experiment. With this in mind, the Journal of Physics D: Applied Physics presents a special roadmap on the correlative microscopy techniques, giving a comprehensive overview from various leading scientists in this field, via a collection of multiple short viewpoints.
RESUMEN
Measuring small forces is a major challenge in cell biology. Here we improve the spatial resolution and accuracy of force reconstruction of the well-established technique of traction force microscopy (TFM) using STED microscopy. The increased spatial resolution of STED-TFM (STFM) allows a greater than 5-fold higher sampling of the forces generated by the cell than conventional TFM, accessing the nano instead of the micron scale. This improvement is highlighted by computer simulations and an activating RBL cell model system.
Asunto(s)
Simulación por Computador , Microscopía de Sonda de Barrido , Modelos Teóricos , Tracción , Algoritmos , Adhesión Celular , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Microscopía de Sonda de Barrido/instrumentación , Microscopía de Sonda de Barrido/métodos , Estrés MecánicoRESUMEN
Wiskott Aldrich syndrome (WAS), an X-linked immunodeficiency, results from loss-of-function mutations in the human hematopoietic cytoskeletal regulator gene WAS. Many missense mutations in the Ena Vasp homology1 (EVH1) domain preserve low-level WAS protein (WASp) expression and confer a milder clinical phenotype. Although disrupted binding to WASp-interacting protein (WIP) leads to enhanced WASp degradation in vivo, the intrinsic function of EVH1-mutated WASp is poorly understood. In the present study, we show that, despite mediating enhanced actin polymerization compared with wild-type WASp in vitro, EVH1 missense mutated proteins did not support full biologic function in cells, even when levels were restored by forced overexpression. Podosome assembly was aberrant and associated with dysregulated lamellipodia formation and impaired persistence of migration. At sites of residual podosome-associated actin polymerization, localization of EVH1-mutated proteins was preserved even after deletion of the entire domain, implying that WIP-WASp complex formation is not absolutely required for WASp localization. However, retention of mutant proteins in podosomes was significantly impaired and associated with reduced levels of WASp tyrosine phosphorylation. Our results indicate that the EVH1 domain is important not only for WASp stability, but also for intrinsic biologic activity in vivo.
Asunto(s)
Células Dendríticas/patología , Mutación Missense , Proteína del Síndrome de Wiskott-Aldrich/genética , Actinas/metabolismo , Animales , Biopolímeros , Proteínas Portadoras/metabolismo , Movimiento Celular , Células Cultivadas , Proteínas del Citoesqueleto , Células Dendríticas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosforilación , Polimerizacion , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Estructura Terciaria de Proteína , Seudópodos/patología , Proteínas Recombinantes de Fusión/fisiología , Eliminación de Secuencia , Organismos Libres de Patógenos Específicos , Proteína del Síndrome de Wiskott-Aldrich/química , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/fisiologíaRESUMEN
Proteins of the ERM family (ezrin, moesin, radixin) play a fundamental role in tethering the membrane to the cellular actin cortex as well as regulating cortical organization and mechanics. Overexpression of dominant inactive forms of ezrin leads to fragilization of the membrane-cortex link and depletion of moesin results in softer cortices that disrupt spindle orientation during cytokinesis. Therefore, the kinetics of association of ERM proteins with the cortex likely influence the timescale of cortical signaling events and the dynamics of membrane interfacing to the cortex. However, little is known about ERM protein turnover at the membrane-cortex interface. Here, we examined cortical ezrin dynamics using fluorescence recovery after photobleaching experiments and single-molecule imaging. Using multiexponential fitting of fluorescence recovery curves, we showed that ezrin turnover resulted from three molecular mechanisms acting on very different timescales. The fastest turnover process was due to association/dissociation from the F-actin cortex, suggesting that ezrin acts as a link that leads to low friction between the membrane and the cortex. The second turnover process resulted from association/dissociation of ezrin from the membrane and the slowest turnover process resulted from the slow diffusion of ezrin in the plane of the membrane. In summary, ezrin-mediated membrane-cortex tethering resulted from long-lived interactions with the membrane via the FERM domain coupled with shorter-lived interactions with the cortex. The slow diffusion of membranous ezrin and its interaction partners relative to the cortex signified that signals emanating from membrane-associated ezrin may locally act to modulate cortical organization and contractility.
Asunto(s)
Actinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Difusión , Transferencia Resonante de Energía de Fluorescencia , Humanos , Cinética , Imagen Molecular , Mutación Puntual , Estabilidad ProteicaRESUMEN
The cytoplasm is the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape changes can occur. Recent experimental evidence suggests that cytoplasmic rheology can be described by a poroelastic model, in which the cytoplasm is treated as a biphasic material consisting of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathed in an interstitial fluid (cytosol). In this picture, the rate of cellular deformation is limited by the rate at which intracellular water can redistribute within the cytoplasm. However, direct supporting evidence for the model is lacking. Here we directly validate the poroelastic model to explain cellular rheology at short timescales using microindentation tests in conjunction with mechanical, chemical and genetic treatments. Our results show that water redistribution through the solid phase of the cytoplasm (cytoskeleton and macromolecular crowders) plays a fundamental role in setting cellular rheology at short timescales.
Asunto(s)
Citoplasma/fisiología , Modelos Biológicos , Fenómenos Biomecánicos , Forma de la Célula , Tamaño de la Célula , Citoesqueleto/fisiología , Elasticidad , Porosidad , Reología , Estrés MecánicoRESUMEN
Cells crucially rely on the interactions of biomolecules at their plasma membrane to maintain homeostasis. Yet, a methodology to systematically quantify biomolecular organisation, measuring diffusion dynamics and oligomerisation, represents an unmet need. Here, we introduce the brightness-transit statistics (BTS) method based on fluorescence fluctuation spectroscopy and combine information from brightness and transit times to elucidate biomolecular diffusion and oligomerisation in both cell-free in vitro and in vitro systems incorporating living cells. We validate our approach in silico with computer simulations and experimentally using oligomerisation of EGFP tethered to supported lipid bilayers. We apply our pipeline to study the oligomerisation of CD40 ectodomain in vitro and endogenous CD40 on primary B cells. While we find a potential for CD40 to oligomerize in a concentration or ligand depended manner, we do not observe mobile oligomers on B cells. The BTS method combines sensitive analysis, quantification, and intuitive visualisation of dynamic biomolecular organisation.
Asunto(s)
Membrana Celular , Proteínas Fluorescentes Verdes , Membrana Dobles de Lípidos , Membrana Celular/metabolismo , Membrana Celular/química , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Humanos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/química , Difusión , Espectrometría de Fluorescencia/métodos , Linfocitos B/metabolismo , Simulación por Computador , Multimerización de Proteína , AnimalesRESUMEN
Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8+ T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4+ and CD8+ T cells enhance CD8+ T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8+ T-cell infiltration. We further show that SEMA3A affects CD8+ T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8+ T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Animales , Humanos , Actinas , Linfocitos T CD8-positivos , Citoesqueleto , Semaforina-3A/genéticaRESUMEN
Immune cells rely on the generation of mechanical force to carry out their function. Consequently, there is a pressing need for quantitative methodologies that permit the probing of the spatio-temporal distribution of mechanical forces generated by immune cells. In this chapter, we provide a guide to quantify immune cell force generation using traction force microscopy (TFM), with a specific focus on its application to the study of the T-cell immunological synapse.
Asunto(s)
Fenómenos Mecánicos , Tracción , Microscopía de Fuerza Atómica/métodos , Sinapsis InmunológicasRESUMEN
Neutrophils are innate immune cells that are key to protecting the host against infection and maintaining body homeostasis. However, if dysregulated, they can contribute to disease, such as in cancer or chronic autoinflammatory disorders. Recent studies have highlighted the heterogeneity in the neutrophil compartment and identified the presence of immature neutrophils and their precursors in these pathologies. Therefore, understanding neutrophil maturity and the mechanisms through which they contribute to disease is critical. Neutrophils were first characterized morphologically by Ehrlich in 1879 using microscopy, and since then, different technologies have been used to assess neutrophil maturity. The advances in the imaging field, including state-of-the-art microscopy and machine learning algorithms for image analysis, reinforce the use of neutrophil nuclear morphology as a fundamental marker of maturity, applicable for objective classification in clinical diagnostics. New emerging approaches, such as the capture of changes in chromatin topology, will provide mechanistic links between the nuclear shape, chromatin organization, and transcriptional regulation during neutrophil maturation.
Asunto(s)
Cromatina , Neutrófilos , Regulación de la Expresión GénicaRESUMEN
Interest in MHC-E-restricted CD8+ T cell responses has been aroused by the discovery of their efficacy in controlling simian immunodeficiency virus (SIV) infection in a vaccine model. The development of vaccines and immunotherapies utilizing human MHC-E (HLA-E)-restricted CD8+ T cell response requires an understanding of the pathway(s) of HLA-E transport and antigen presentation, which have not been clearly defined previously. We show here that, unlike classical HLA class I, which rapidly exits the endoplasmic reticulum (ER) after synthesis, HLA-E is largely retained because of a limited supply of high-affinity peptides, with further fine-tuning by its cytoplasmic tail. Once at the cell surface, HLA-E is unstable and is rapidly internalized. The cytoplasmic tail plays a crucial role in facilitating HLA-E internalization, which results in its enrichment in late and recycling endosomes. Our data reveal distinctive transport patterns and delicate regulatory mechanisms of HLA-E, which help to explain its unusual immunological functions.
Asunto(s)
Antígenos de Histocompatibilidad Clase I , Vacunas , Animales , Humanos , Antígenos de Histocompatibilidad Clase I/metabolismo , Linfocitos T CD8-positivos , Presentación de Antígeno , Antígenos HLA-ERESUMEN
Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single-plane image acquisition within a subcellular-sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of living cells.