Exploring Mechanical Forces Shaping Self-Organization and Morphogenesis During Early Embryo Development.

Embryonic development is a dynamic process orchestrated by a delicate interplay of biochemical and biophysical factors. While the role of genetics and biochemistry in embryogenesis has been extensively studied, recent research has highlighted the significance of mechanical regulation in shaping and guiding this intricate process. Here, we provide an overview of the current understanding of the mechanical regulation of embryo development. We explore how mechanical forces generated by cells and tissues play a crucial role in driving the development of different stages. We examine key morphogenetic processes such as compaction, blastocyst formation, implantation, and egg cylinder formation, and discuss the mechanical mechanisms and cues involved. By synthesizing the current body of literature, we highlight the emerging concepts and open questions in the field of mechanical regulation. We aim to provide an overview of the field, inspiring future investigations and fostering a deeper understanding of the mechanical aspects of embryo development.

Mesenchymal Vangl1 and Vangl2 facilitate airway elongation and widening independently of the planar cell polarity complex.

Adult mammalian lungs exhibit a fractal pattern, as each successive generation of airways is a fraction of the size of the parental branch. Achieving this structure likely requires precise control of airway length and diameter, as the embryonic airways initially lack the fractal scaling observed in the adult. In monolayers and tubes, directional growth can be regulated by the planar cell polarity (PCP) complex. Here, we characterized the roles of PCP complex components in airway initiation, elongation and widening during branching morphogenesis of the lung. Using tissue-specific knockout mice, we surprisingly found that branching morphogenesis proceeds independently of PCP complex function in the lung epithelium. Instead, we found a previously unreported Celsr1-independent role for the PCP complex components Vangl1 and Vangl2 in the pulmonary mesenchyme, where they are required for branch initiation, elongation and widening. Our data thus reveal an explicit function for Vangl1 and Vangl2 that is independent of the core PCP complex, suggesting a functional diversification of PCP complex components in vertebrate development. These data also reveal an essential role for the embryonic mesenchyme in generating the fractal structure of airways in the mature lung.

Actomyosin contraction in the follicular epithelium provides the major mechanical force for follicle rupture during Drosophila ovulation.

Ovulation is critical for sexual reproduction and consists of the process of liberating fertilizable oocytes from their somatic follicle capsules, also known as follicle rupture. The mechanical force for oocyte expulsion is largely unknown in many species. Our previous work demonstrated that Drosophila ovulation, as in mammals, requires the proteolytic degradation of the posterior follicle wall and follicle rupture to release the mature oocyte from a layer of somatic follicle cells. Here, we identified actomyosin contraction in somatic follicle cells as the major mechanical force for follicle rupture. Filamentous actin (F-actin) and nonmuscle myosin II (NMII) are highly enriched in the cortex of follicle cells upon stimulation with octopamine (OA), a monoamine critical for Drosophila ovulation. Pharmacological disruption of F-actin polymerization prevented follicle rupture without interfering with the follicle wall breakdown. In addition, we demonstrated that OA induces Rho1 guanosine triphosphate (GTP)ase activation in the follicle cell cortex, which activates Ras homolog (Rho) kinase to promote actomyosin contraction and follicle rupture. All these results led us to conclude that OA signaling induces actomyosin cortex enrichment and contractility, which generates the mechanical force for follicle rupture during Drosophila ovulation. Due to the conserved nature of actomyosin contraction, this work could shed light on the mechanical force required for follicle rupture in other species including humans.

Mechanical force matters in early T cell activation.

Mechanical force has repeatedly been highlighted to be involved in T cell activation. However, the biological significance of mechanical force for T cell receptor signaling remains under active consideration. Here, guided by theoretical analysis, we provide a perspective on how mechanical forces between a T cell and an antigen-presenting cell can influence the bond of a single T cell receptor major histocompatibility complex during early T cell activation. We point out that the lifetime of T cell receptor bonds and thus the degree of their phosphorylation which is essential for T cell activation depends considerably on the T cell receptor rigidity and the average magnitude and frequency of an applied oscillatory force. Such forces could be, for example, produced by protrusions like microvilli during early T cell activation or invadosomes during full T cell activation. These features are suggestive of mechanical force being exploited by T cells to advance self-nonself discrimination in early T cell activation.

Force: A messenger of axon outgrowth.

The axon is a sophisticated macromolecular machine composed of interrelated parts that transmit signals like spur gears transfer motion between parallel shafts. The growth cone is a fine sensor that integrates mechanical and chemical cues and transduces these signals through the generation of a traction force that pushes the tip and pulls the axon shaft forward. The axon shaft, in turn, senses this pulling force and transduces this signal in an orchestrated response, coordinating cytoskeleton remodeling and intercalated mass addition to sustain and support the advancing of the tip. Extensive research suggests that the direct application of active force is per se a powerful inducer of axon growth, potentially bypassing the contribution of the growth cone. This review provides a critical perspective on current knowledge of how the force is a messenger of axon growth and its mode of action for controlling navigation, including aspects that remain unclear. It also focuses on novel approaches and tools designed to mechanically manipulate axons, and discusses their implications in terms of potential novel therapies for re-wiring the nervous system.

Exploring the therapeutic potential of urine-derived stem cell exosomes in temporomandibular joint osteoarthritis.

Temporomandibular joint osteoarthritis (TMJOA) is a degenerative ailment that causes slow cartilage degeneration, aberrant bone remodeling, and persistent discomfort, leading to a considerable reduction in the patient's life quality. Current treatment options for TMJOA have limited efficacy. This investigation aimed to explore a potential strategy for halting or reversing the progression of TMJOA through the utilization of exosomes (EXOs) derived from urine-derived stem cells (USCs). The USC-EXOs were obtained through microfiltration and ultrafiltration techniques, followed by their characterization using particle size analysis, electron microscopy, and immunoblotting. Subsequently, an in vivo model of TMJOA induced by mechanical force was established. To assess the changes in the cartilage of TMJOA treated with USC-EXOs, we performed histology analysis using hematoxylin-eosin staining, immunohistochemistry, and histological scoring. Our findings indicate that the utilization of USC-EXOs yields substantial reductions in TMJOA, while concurrently enhancing the structural integrity and smoothness of the compromised condylar cartilage surface. Additionally, USC-EXOs exhibit inhibitory effects on osteoclastogenic activity within the subchondral bone layer of the condylar cartilage, as well as attenuated apoptosis in the rat TMJ in response to mechanical injury. In conclusion, USC-EXOs hold considerable promise as a potential therapeutic intervention for TMJOA.

Mechanical force determines chimeric antigen receptor microclustering and signaling.

Chimeric antigen receptor (CAR) T cells are activated to trigger the lytic machinery after antigen engagement, and this has been successfully applied clinically as therapy. The mechanism by which antigen binding leads to the initiation of CAR signaling remains poorly understood. Here, we used a set of short double-stranded DNA (dsDNA) tethers with mechanical forces ranging from ∼12 to ∼51 pN to manipulate the mechanical force of antigen tether and decouple the microclustering and signaling events. Our results revealed that antigen-binding-induced CAR microclustering and signaling are mechanical force dependent. Additionally, the mechanical force delivered to the antigen tether by the CAR for microclustering is generated by autonomous cell contractility. Mechanistically, the mechanical-force-induced strong adhesion and CAR diffusion confinement led to CAR microclustering. Moreover, cytotoxicity may have a lower mechanical force threshold than cytokine generation. Collectively, these results support a model of mechanical-force-induced CAR microclustering for signaling.

Proteomic and Phosphoproteomic Profiling of Matrix Stiffness-Induced Stemness-Dormancy State Transition in Breast Cancer Cells.

The dormancy of cancer stem cells is a major factor leading to drug resistance and a high rate of late recurrence and mortality in estrogen receptor-positive (ER+) breast cancer. Previously, we demonstrated that a stiffer matrix induces tumor cell dormancy and drug resistance, whereas a softened matrix promotes tumor cells to exhibit a stem cell state with high proliferation and migration. In this study, we present a comprehensive analysis of the proteome and phosphoproteome in response to gradient changes in matrix stiffness, elucidating the mechanisms behind cell dormancy-induced drug resistance. Overall, we found that antiapoptotic and membrane transport processes may be involved in the mechanical force-induced dormancy resistance of ER+ breast cancer cells. Our research provides new insights from a holistic proteomic and phosphoproteomic perspective, underscoring the significant role of mechanical forces stemming from the stiffness of the surrounding extracellular matrix as a critical regulatory factor in the tumor microenvironment.

GPX4-mediated bone ferroptosis under mechanical stress decreased bone formation via the YAP-TEAD signalling pathway.

Fracture of the alveolar bone resorption is a common complication in orthodontic treatment, which mainly caused by extreme mechanical loading. However, the ferroptosis with orthodontic tooth movement(OTM) relationship has not been thoroughly described. We here analysed whether ferroptosis is involved in OTM-associated alveolar bone loss. Mouse osteoblasts (MC-3T3) and knockdown glutathione peroxidase 4 (GPX4) MC-3T3 were stimulated with compressive force loading and ferrostatin-1 (Fer-1, a ferroptosis inhibitor), and the changes in lipid peroxidation morphology, expression of ferroptosis-related factors and osteogenesis levels were detected. After establishing the rat experimental OTM model, the changes in ferroptosis-related factors and osteogenesis levels were reevaluated in the same manner. Ferroptosis was involved in mechanical stress regulating osteoblast remodelling, and Fer-1 and erastin affected osteoblasts under compression force loading. Fer-1 regulated ferroptosis and autophagy in MC-3T3 and promoted bone proliferation. GPX4-dependent ferroptosis stimulated the YAP (homologous oncoproteins Yes-associated protein) pathway, and GPX4 promoted ferroptosis via the YAP-TEAD (transcriptional enhanced associate domain) signal pathway under mechanical compression force. The in vivo experiment results were consistent with the in vitro experiment results. Ferroptosis transpires during the motion of orthodontic teeth, with compression force side occurring earlier than stretch side within 4 h. GPX4 plays an important role in alveolar bone loss, while Fer-1 can inhibit the compression force-side alveolar bone loss. GPX4's Hippo-YAP pathway is activated by the lack of compression force in the lateral alveolar bone.

Macrophage M2 polarization promotes pulpal inflammation resolution during orthodontic tooth movement.

Mechanical force induces hypoxia in the pulpal area by compressing the apical blood vessels of the pulp, triggering pulpal inflammation during orthodontic tooth movement. However, this inflammation tends to be restorable. Macrophages are recognized as pivotal immunoreactive cells in the dental pulp. Whether they are involved in the resolution of pulpal inflammation in orthodontic teeth remains unclear. In this study, we investigated macrophage polarization and its effects during orthodontic tooth movement. It was demonstrated that macrophages within the dental pulp polarized to M2 type and actively participated in the process of pulpal inflammation resolution. Inflammatory reactions were generated and vascularization occurred in the pulp during orthodontic tooth movement. Macrophages in orthodontic pulp show a tendency to polarize towards M2 type as a result of pulpal hypoxia. Furthermore, by blocking M2 polarization, we found that macrophage M2 polarization inhibits dental pulp-secreting inflammatory factors and enhances VEGF production. In conclusion, our findings suggest that macrophages promote pulpal inflammation resolution by enhancing M2 polarization and maintaining dental health during orthodontic tooth movement.

A Simply Synthesized Shaking-induced Small Molecule System with Repeatable and Instantaneous Discoloration Response.

Force-related discoloration materials are highly valuable because of their characteristics of visualization, easy operation, and environment friendliness. Most force-related discoloration materials focus on polymers and depend on bond scission, which leads to insensitivity and unrecoverable. Small-molecule systems based on well-defined molecular structures and simple composition with high sensitivity would exhibit considerable mechanochromic potential. However, to date, researches about force-related discoloration materials based on small molecule solution remain limited and are rarely reported. In this study, we developed a repeatable and instantaneous discoloration small molecule solution system by simple one-pot synthesis method. It exhibited an instantaneous chromic change from yellowish to dark green under shaking and reverting back to yellow within 1 minute after removal of the shaking. Experimental results confirmed that the discoloration mechanism is attributed to the oscillation accelerating the production of unstable ortho-OH phenoxyl radical. The newly developed shaking-induced discoloration small molecule system (SDSMS) promises in field of mechanical force sensing and optical encryption.

Ubiquitin C-terminal hydrolase L1 activation in periodontal ligament cells mediates orthodontic tooth movement via the MAPK signaling pathway.

PURPOSE: Periodontal ligament cells (PDLCs) play a significant role in orthodontic force induced bone remodeling. However, the molecular mechanisms by which PDLCs respond to mechanical stimuli and influence osteoclastic activities remain unclear. This study aims to investigate the role of UCHL1, a key deubiquitinating enzyme involved in protein degradation and cellular responses, in force-treated PDLCs during orthodontic tooth movement (OTM). MATERIALS AND METHODS: In this study, we conducted in vivo and in vitro experiments using human PDLCs and a rat model of OTM. Mechanical stress was applied to PDLCs, and UCHL1 expression was analyzed through quantitative real-time polymerase chain reaction (qPCR), Western blot, and immunofluorescence staining. UCHL1 knockdown was achieved using siRNA, and its effects on osteoclast differentiation were assessed. The role of the MAPK/ERK pathway was investigated using the MEK-specific inhibitor U0126. An animal model of OTM was established, and the impact of UCHL1 inhibitor-LDN57444 on OTM and osteoclastic activity was evaluated through micro-CT analysis, histological staining, and immunohistochemistry. RESULTS: Mechanical force induced UCHL1 expression in PDLCs during OTM. UCHL1 knockdown downregulated the RANKL/OPG ratio in PDLCs, affecting osteoclast differentiation. LDN57444 inhibited OTM and osteoclastic activity. UCHL1 activation correlated with ERK1/2 phosphorylation in force-treated PDLCs. CONCLUSIONS: Mechanical force mediated UCHL1 activation in PDLCs promotes osteoclast differentiation via the ERK1/2 signaling pathway during OTM.

Intertissue mechanical interactions shape the olfactory circuit in zebrafish.

While the chemical signals guiding neuronal migration and axon elongation have been extensively studied, the influence of mechanical cues on these processes remains poorly studied in vivo. Here, we investigate how mechanical forces exerted by surrounding tissues steer neuronal movements and axon extension during the morphogenesis of the olfactory placode in zebrafish. We mainly focus on the mechanical contribution of the adjacent eye tissue, which develops underneath the placode through extensive evagination and invagination movements. Using quantitative analysis of cell movements and biomechanical manipulations, we show that the developing eye exerts lateral traction forces on the olfactory placode through extracellular matrix, mediating proper morphogenetic movements and axon extension within the placode. Our data shed new light on the key participation of intertissue mechanical interactions in the sculpting of neuronal circuits.

Immune cells fold and damage fungal hyphae.

Innate immunity provides essential protection against life-threatening fungal infections. However, the outcomes of individual skirmishes between immune cells and fungal pathogens are not a foregone conclusion because some pathogens have evolved mechanisms to evade phagocytic recognition, engulfment, and killing. For example, Candida albicans can escape phagocytosis by activating cellular morphogenesis to form lengthy hyphae that are challenging to engulf. Through live imaging of C. albicans-macrophage interactions, we discovered that macrophages can counteract this by folding fungal hyphae. The folding of fungal hyphae is promoted by Dectin-1, ß2-integrin, VASP, actin-myosin polymerization, and cell motility. Folding facilitates the complete engulfment of long hyphae in some cases and it inhibits hyphal growth, presumably tipping the balance toward successful fungal clearance.

The Role of Mechanotransduction in Contact Inhibition of Locomotion and Proliferation.

Contact inhibition (CI) represents a crucial tumor-suppressive mechanism responsible for controlling the unbridled growth of cells, thus preventing the formation of cancerous tissues. CI can be further categorized into two distinct yet interrelated components: CI of locomotion (CIL) and CI of proliferation (CIP). These two components of CI have historically been viewed as separate processes, but emerging research suggests that they may be regulated by both distinct and shared pathways. Specifically, recent studies have indicated that both CIP and CIL utilize mechanotransduction pathways, a process that involves cells sensing and responding to mechanical forces. This review article describes the role of mechanotransduction in CI, shedding light on how mechanical forces regulate CIL and CIP. Emphasis is placed on filamin A (FLNA)-mediated mechanotransduction, elucidating how FLNA senses mechanical forces and translates them into crucial biochemical signals that regulate cell locomotion and proliferation. In addition to FLNA, trans-acting factors (TAFs), which are proteins or regulatory RNAs capable of directly or indirectly binding to specific DNA sequences in distant genes to regulate gene expression, emerge as sensitive players in both the mechanotransduction and signaling pathways of CI. This article presents methods for identifying these TAF proteins and profiling the associated changes in chromatin structure, offering valuable insights into CI and other biological functions mediated by mechanotransduction. Finally, it addresses unanswered research questions in these fields and delineates their possible future directions.

Compressive force regulates orthodontic tooth movement via activating the NLRP3 inflammasome.

Mechanical stress regulates various cellular functions like cell inflammation, immune responses, proliferation, and differentiation to maintain tissue homeostasis. However, the impact of mechanical signals on macrophages and the underlying mechanisms by which mechanical force regulates bone remodeling during orthodontic tooth movement remain unclear. NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome has been reported to promote osteoclastic differentiation to regulate alveolar bone resorption. But the relationship between the compressive force and NLRP3 inflammasome in macrophages remains unknown. In this study, immunohistochemical staining results showed elevated expression of NLRP3 and interleukin-1ß, as well as an increased number of macrophages expressing NLRP3, on the compression side of the periodontal tissues, after force application for 7 days. Furthermore, the number of tartrate-resistant acid phosphatase-positive osteoclasts, and the mRNA and protein expression levels of osteoclast-related genes in the periodontal tissue decreased in the Nlrp3-/- mice compared to the WT mice group after orthodontic movement. In vitro mechanical force activates the NLRP3 inflammasome and inhibits autophagy. Intraperitoneal injection of the autophagy inhibitor 3-methyladenine in Nlrp3-/- mice promoted orthodontic tooth movement. This result indicates that the absence of NLRP3 inflammasome activation can be partially compensated for by autophagy inhibitors. Mechanistically, force-induced activation of the NLRP3 inflammasome in macrophages via the cGAS/P2X7R axis. In conclusion, compressive force regulates orthodontic tooth movement via activating the NLRP3 inflammasome.

Independent endothelial functions of PIEZO1 and TRPV4 in hepatic portal vein and predominance of PIEZO1 in mechanical and osmotic stress.

BACKGROUND & AIMS: PIEZO1 and TRPV4 are mechanically and osmotically regulated calcium-permeable channels. The aim of this study was to determine the relevance and relationship of these channels in the contractile tone of the hepatic portal vein, which experiences mechanical and osmotic variations as it delivers blood to the liver from the intestines, gallbladder, pancreas and spleen. METHODS: Wall tension was measured in freshly dissected portal veins from adult male mice, which were genetically unmodified or modified for either a non-disruptive tag in native PIEZO1 or endothelial-specific PIEZO1 deletion. Pharmacological agents were used to activate or inhibit PIEZO1, TRPV4 and associated pathways, including Yoda1 and Yoda2 for PIEZO1 and GSK1016790A for TRPV4 agonism, respectively. RESULTS: PIEZO1 activation leads to nitric oxide synthase- and endothelium-dependent relaxation of the portal vein. TRPV4 activation causes contraction, which is also endothelium-dependent but independent of nitric oxide synthase. The TRPV4-mediated contraction is suppressed by inhibitors of phospholipase A2 and cyclooxygenases and mimicked by prostaglandin E2 , suggesting mediation by arachidonic acid metabolism. TRPV4 antagonism inhibits the effect of agonising TRPV4 but not PIEZO1. Increased wall stretch and hypo-osmolality inhibit TRPV4 responses while lacking effects on or amplifying PIEZO1 responses. CONCLUSIONS: The portal vein contains independently functioning PIEZO1 channels and TRPV4 channels in the endothelium, the pharmacological activation of which leads to opposing effects of vessel relaxation (PIEZO1) and contraction (TRPV4). In mechanical and osmotic strain, the PIEZO1 mechanism dominates. Modulators of these channels could present important new opportunities for manipulating liver perfusion and regeneration in disease and surgical procedures.

Mechanical force induces macrophage-derived exosomal UCHL3 promoting bone marrow mesenchymal stem cell osteogenesis by targeting SMAD1.

BACKGROUND: Orthodontic tooth movement (OTM), a process of alveolar bone remodelling, is induced by mechanical force and regulated by local inflammation. Bone marrow-derived mesenchymal stem cells (BMSCs) play a fundamental role in osteogenesis during OTM. Macrophages are mechanosensitive cells that can regulate local inflammatory microenvironment and promote BMSCs osteogenesis by secreting diverse mediators. However, whether and how mechanical force regulates osteogenesis during OTM via macrophage-derived exosomes remains elusive. RESULTS: Mechanical stimulation (MS) promoted bone marrow-derived macrophage (BMDM)-mediated BMSCs osteogenesis. Importantly, when exosomes from mechanically stimulated BMDMs (MS-BMDM-EXOs) were blocked, the pro-osteogenic effect was suppressed. Additionally, compared with exosomes derived from BMDMs (BMDM-EXOs), MS-BMDM-EXOs exhibited a stronger ability to enhance BMSCs osteogenesis. At in vivo, mechanical force-induced alveolar bone formation was impaired during OTM when exosomes were blocked, and MS-BMDM-EXOs were more effective in promoting alveolar bone formation than BMDM-EXOs. Further proteomic analysis revealed that ubiquitin carboxyl-terminal hydrolase isozyme L3 (UCHL3) was enriched in MS-BMDM-EXOs compared with BMDM-EXOs. We went on to show that BMSCs osteogenesis and mechanical force-induced bone formation were impaired when UCHL3 was inhibited. Furthermore, mothers against decapentaplegic homologue 1 (SMAD1) was identified as the target protein of UCHL3. At the mechanistic level, we showed that SMAD1 interacted with UCHL3 in BMSCs and was downregulated when UCHL3 was suppressed. Consistently, overexpression of SMAD1 rescued the adverse effect of inhibiting UCHL3 on BMSCs osteogenesis. CONCLUSIONS: This study suggests that mechanical force-induced macrophage-derived exosomal UCHL3 promotes BMSCs osteogenesis by targeting SMAD1, thereby promoting alveolar bone formation during OTM.

Mechanical force induces mitophagy-mediated anaerobic oxidation in periodontal ligament stem cells.

BACKGROUND: The preference for glucose oxidative mode has crucial impacts on various physiological activities, including determining stem cell fate. External mechanical factors can play a decisive role in regulating critical metabolic enzymes and pathways of stem cells. Periodontal ligament stem cells (PDLSCs) are momentous effector cells that transform mechanical force into biological signals during the reconstruction of alveolar bone. However, mechanical stimuli-induced alteration of oxidative characteristics in PDLSCs and the underlying mechanisms have not been fully elucidated. METHODS: Herein, we examined the expression of LDH and COX4 by qRT-PCR, western blot, immunohistochemistry and immunofluorescence. We detected metabolites of lactic acid and reactive oxygen species for functional tests. We used tetramethylrhodamine methyl ester (TMRM) staining and a transmission electron microscope to clarify the mitochondrial status. After using western blot and immunofluorescence to clarify the change of DRP1, we further examined MFF, PINK1, and PARKIN by western blot. We used cyclosporin A (CsA) to confirm the regulation of mitophagy and ceased the stretching as a rescue experiment. RESULTS: Herein, we ascertained that mechanical force could increase the level of LDH and decrease the expression of COX4 in PDLSCs. Simultaneously, the yield of reactive oxygen species (ROS) in PDLSC reduced after stretching, while lactate acid augmented significantly. Furthermore, mitochondrial function in PDLSCs was negatively affected by impaired mitochondrial membrane potential (MMP) under mechanical force, and the augment of mitochondrial fission further induced PRKN-dependent mitophagy, which was confirmed by the rescue experiments via blocking mitophagy. As a reversible physiological stimulation, the anaerobic preference of PDLSCs altered by mechanical force could restore after the cessation of force stimulation. CONCLUSIONS: Altogether, our study demonstrates that PDLSCs under mechanical force preferred anaerobic oxidation induced by the affected mitochondrial dynamics, especially mitophagy. Our findings support an association between mechanical stimulation and the oxidative profile of stem cells, which may shed light on the mechanical guidance of stem cell maintenance and commitment, and lay a molecular foundation for periodontal tissue regeneration.

Co-translational folding of nascent polypeptides: Multi-layered mechanisms for the efficient biogenesis of functional proteins.

The coupling of protein synthesis and folding is a crucial yet poorly understood aspect of cellular protein folding. Over the past few years, it has become possible to experimentally follow and define protein folding on the ribosome, revealing principles that shape co-translational folding and distinguish it from refolding in solution. Here, we highlight some of these recent findings from biochemical and biophysical studies and their potential significance for cellular protein biogenesis. In particular, we focus on nascent chain interactions with the ribosome, interactions within the nascent protein, modulation of translation elongation rates, and the role of mechanical force that accompanies nascent protein folding. The ability to obtain mechanistic insight in molecular detail has set the stage for exploring the intricate process of nascent protein folding. We believe that the aspects discussed here will be generally important for understanding how protein synthesis and folding are coupled and regulated.