Methods for assessment of membrane protrusion dynamics.

Membrane protrusions are a critical facet of cell function. Mediating fundamental processes such as cell migration, cell-cell interactions, phagocytosis, as well as assessment and remodeling of the cell environment. Different protrusion types and morphologies can promote different cellular functions and occur downstream of distinct signaling pathways. As such, techniques to quantify and understand the inner workings of protrusion dynamics are critical for a comprehensive understanding of cell biology. In this chapter, we describe approaches to analyze cellular protrusions and correlate physical changes in cell morphology with biochemical signaling processes. We address methods to quantify and characterize protrusion types and velocity, mathematical approaches to predictive models of cytoskeletal changes, and implementation of protein engineering and biosensor design to dissect cell signaling driving protrusive activity. Combining these approaches allows cell biologists to develop a comprehensive understanding of the dynamics of membrane protrusions.

A role for Mitochondrial Rho GTPase 1 (MIRO1) in motility and membrane dynamics of peroxisomes.

Peroxisomes are dynamic organelles which fulfil essential roles in lipid and ROS metabolism. Peroxisome movement and positioning allows interaction with other organelles and is crucial for their cellular function. In mammalian cells, such movement is microtubule-dependent and mediated by kinesin and dynein motors. The mechanisms of motor recruitment to peroxisomes are largely unknown, as well as the role this plays in peroxisome membrane dynamics and proliferation. Here, using a combination of microscopy, live-cell imaging analysis and mathematical modelling, we identify a role for Mitochondrial Rho GTPase 1 (MIRO1) as an adaptor for microtubule-dependent peroxisome motility in mammalian cells. We show that MIRO1 is targeted to peroxisomes and alters their distribution and motility. Using a peroxisome-targeted MIRO1 fusion protein, we demonstrate that MIRO1-mediated pulling forces contribute to peroxisome membrane elongation and proliferation in cellular models of peroxisome disease. Our findings reveal a molecular mechanism for establishing peroxisome-motor protein associations in mammalian cells and provide new insights into peroxisome membrane dynamics in health and disease.

Actin assembly mechanisms at a glance.

The actin cytoskeleton and associated motor proteins provide the driving forces for establishing the astonishing morphological diversity and dynamics of mammalian cells. Aside from functions in protruding and contracting cell membranes for motility, differentiation or cell division, the actin cytoskeleton provides forces to shape and move intracellular membranes of organelles and vesicles. To establish the many different actin assembly functions required in time and space, actin nucleators are targeted to specific subcellular compartments, thereby restricting the generation of specific actin filament structures to those sites. Recent research has revealed that targeting and activation of actin filament nucleators, elongators and myosin motors are tightly coordinated by conserved protein complexes to orchestrate force generation. In this Cell Science at a Glance article and the accompanying poster, we summarize and discuss the current knowledge on the corresponding protein complexes and their modes of action in actin nucleation, elongation and force generation.

Mechanisms of collective cell movement lacking a leading or free front edge in vivo.

Collective cell movement is one of the strategies for achieving the complex shapes of tissues and organs. In this process, multiple cells within a group held together by cell-cell adhesion acquire mobility and move together in the same direction. In some well-studied models of collective cell movement, the mobility depends strongly on traction generated at the leading edge by cells located at the front. However, recent advances in live-imaging techniques have led to the discovery of other types of collective cell movement lacking a leading edge or even a free edge at the front, in a diverse array of morphological events, including tubule elongation, epithelial sheet extension, and tissue rotation. We herein review some of the developmental events that are organized by collective cell movement and attempt to elucidate the underlying cellular and molecular mechanisms, which include membrane protrusions, guidance cues, cell intercalation, and planer cell polarity, or chirality pathways.

Cooperative interactions of LPPR family members in membrane localization and alteration of cellular morphology.

The lipid phosphate phosphatase-related proteins (LPPRs), also known as plasticity-related genes (PRGs), are classified as a new brain-enriched subclass of the lipid phosphate phosphatase (LPP) superfamily. They induce membrane protrusions, neurite outgrowth or dendritic spine formation in cell lines and primary neurons. However, the exact roles of LPPRs and the mechanisms underlying their effects are not certain. Here, we present the results of a large-scale proteome analysis to determine LPPR1-interacting proteins using co-immunoprecipitation coupled to mass spectrometry. We identified putative LPPR1-binding proteins involved in various biological processes. Most interestingly, we identified the interaction of LPPR1 with its family member LPPR3, LPPR4 and LPPR5. Their interactions were characterized by co-immunoprecipitation and colocalization analysis using confocal and super-resolution microscopy. Moreover, co-expressing two LPPR members mutually elevated their protein levels, facilitated their plasma membrane localization and resulted in an increased induction of membrane protrusions as well as the phosphorylation of S6 ribosomal protein. Taken together, we revealed a new functional cooperation between LPPR family members and discovered for the first time that LPPRs likely exert their function through forming complex with its family members.

The growth determinants and transport properties of tunneling nanotube networks between B lymphocytes.

Tunneling nanotubes (TNTs) are long intercellular connecting structures providing a special transport route between two neighboring cells. To date TNTs have been reported in different cell types including immune cells such as T-, NK, dendritic cells, or macrophages. Here we report that mature, but not immature, B cells spontaneously form extensive TNT networks under conditions resembling the physiological environment. Live-cell fluorescence, structured illumination, and atomic force microscopic imaging provide new insights into the structure and dynamics of B cell TNTs. Importantly, the selective interaction of cell surface integrins with fibronectin or laminin extracellular matrix proteins proved to be essential for initiating TNT growth in B cells. These TNTs display diversity in length and thickness and contain not only F-actin, but their majority also contain microtubules, which were found, however, not essential for TNT formation. Furthermore, we demonstrate that Ca2+-dependent cortical actin dynamics exert a fundamental control over TNT growth-retraction equilibrium, suggesting that actin filaments form the TNT skeleton. Non-muscle myosin 2 motor activity was shown to provide a negative control limiting the uncontrolled outgrowth of membranous protrusions. Moreover, we also show that spontaneous growth of TNTs is either reduced or increased by B cell receptor- or LPS-mediated activation signals, respectively, thus supporting the critical role of cytoplasmic Ca2+ in regulation of TNT formation. Finally, we observed transport of various GM1/GM3+ vesicles, lysosomes, and mitochondria inside TNTs, as well as intercellular exchange of MHC-II and B7-2 (CD86) molecules which may represent novel pathways of intercellular communication and immunoregulation.

Myosin light chain kinase (MLCK) regulates cell migration in a myosin regulatory light chain phosphorylation-independent mechanism.

Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.

SH2B1 increases the numbers of IRSp53-induced filopodia.

BACKGROUND: Filopodia are actin-rich membrane protrusions that play instrumental roles in development, cell migration, pathogen detection, and wound healing. During neurogenesis, filopodium formation precedes the formation of dendrites and spines. The insulin receptor substrate protein of 53kDa (IRSp53) has been implicated in regulating the formation of filopodia. Our previous results suggest that a signaling adaptor protein SH2B1ß is required for neurite outgrowth of hippocampal neurons and neurite initiation of PC12 cells. Thus, we hypothesize that IRSp53 and SH2B1ß may act together to regulate filopodium formation. METHODS: To determine the contribution of IRSp53 and SH2B1ß in the formation of filopodia, we transiently transfect IRSp53 and/or SH2B1ß to 293T cells. Cell morphology and protein distribution are assessed via confocal microscopy and subcellular fractionation. Total numbers of filopodia and filopodium numbers per perimeter are calculated to show the relative contribution of IRSp53 and SH2B1ß. RESULTS: In this study, we show that SH2B1ß interacts with IRSp53 and increases the number of IRSp53-induced filopodia. One mechanism for this enhancement is that IRSp53 recruits SH2B1ß to the plasma membrane to actively promote membrane protrusion. The increased numbers of filopodia likely result from SH2B1-mediated cytoplasmic extension and thus increased cell perimeter as well as IRSp53-mediated filopodium formation. CONCLUSIONS: Taken together, this study provides a novel finding that SH2B1ß interacts with IRSp53-containing complexes to increase the number of filopodia. GENERAL SIGNIFICANCE: A better understanding of how SH2B1ß and IRSp53 promote filopodium formation may have clinical implication in neurogenesis and regeneration.

MafB antagonizes phenotypic alteration induced by GM-CSF in microglia.

Microglia are tissue-resident macrophages which are distributed throughout the central nervous system (CNS). Recent studies suggest that microglia are a unique myeloid population distinct from peripheral macrophages in terms of origin and gene expression signature. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a pleiotropic cytokine regulating myeloid development, has been shown to stimulate proliferation and alter phenotype of microglia in vitro. However, how its signaling is modulated in microglia is poorly characterized. MafB, a bZip transcriptional factor, is highly expressed in monocyte-macrophage lineage cells including microglia, although its role in microglia is largely unknown. We investigated the crosstalk between GM-CSF signaling and MafB by analyzing primary microglia. We found that Mafb-deficient microglia grew more rapidly than wild-type microglia in response to GM-CSF. Moreover, the expression of genes associated with microglial differentiation was more downregulated in Mafb-deficient microglia cultured with GM-CSF. Notably, such differences between the genotypes were not observed in the presence of M-CSF. In addition, we found that Mafb-deficient microglia cultured with GM-CSF barely extended their membrane protrusions, probably due to abnormal activation of RhoA, a key regulator of cytoskeletal remodeling. Altogether, our study reveals that MafB is a negative regulator of GM-CSF signaling in microglia. These findings could provide new insight into the modulation of cytokine signaling by transcription factors in microglia.

The WAVE complex forms linear arrays at negative membrane curvature to instruct lamellipodia formation.

Cells generate a wide range of actin-based membrane protrusions for various cell behaviors. These protrusions are organized by different actin nucleation promoting factors. For example, N-WASP controls finger-like filopodia, whereas the WAVE complex controls sheet-like lamellipodia. These different membrane morphologies likely reflect different patterns of nucleator self-organization. N-WASP phase separation has been successfully studied through biochemical reconstitutions, but how the WAVE complex self-organizes to instruct lamellipodia is unknown. Because WAVE complex self-organization has proven refractory to cell-free studies, we leverage in vivo biochemical approaches to investigate WAVE complex organization within its native cellular context. With single molecule tracking and molecular counting, we show that the WAVE complex forms highly regular multilayered linear arrays at the plasma membrane that are reminiscent of a microtubule-like organization. Similar to the organization of microtubule protofilaments in a curved array, membrane curvature is both necessary and sufficient for formation of these WAVE complex linear arrays, though actin polymerization is not. This dependency on negative membrane curvature could explain both the templating of lamellipodia and their emergent behaviors, including barrier avoidance. Our data uncover the key biophysical properties of mesoscale WAVE complex patterning and highlight an integral relationship between NPF self-organization and cell morphogenesis.

Localization of host endocytic and actin-associated proteins during Shigella flexneri intracellular motility and intercellular spreading.

Shigella flexneri (S. flexneri), the causative agent of bacillary dysentery, uses an effector-mediated strategy to hijack host cells and cause disease. To propagate and spread within human tissues, S. flexneri bacteria commandeer the host actin cytoskeleton to generate slender actin-rich comet tails to move intracellularly, and later, plasma membrane actin-based protrusions to move directly between adjacent host cells. To facilitate intercellular bacterial spreading, large micron-sized endocytic-like membrane invaginations form at the periphery of neighboring host cells that come into contact with S. flexneri-containing membrane protrusions. While S. flexneri comet tails and membrane protrusions consist primarily of host actin cytoskeletal proteins, S. flexneri membrane invaginations remain poorly understood with only clathrin and the clathrin adapter epsin-1 localized to the structures. Tangentially, we recently reported that Listeria monocytogenes, another actin-hijacking pathogen, exploits an assortment of caveolar and actin-bundling proteins at their micron-sized membrane invaginations formed during their cell-to-cell movement. Thus, to further characterize the S. flexneri disease process, we set out to catalog the distribution of a variety of actin-associated and caveolar proteins during S. flexneri actin-based motility and cell-to-cell spreading. Here we show that actin-associated proteins found at L. monocytogenes comet tails and membrane protrusions mimic those present at S. flexneri comet tails with the exception of α-actinins 1 and 4, which were shed from S. flexneri membrane protrusions. We also demonstrate that all known host endocytic components found at L. monocytogenes membrane invaginations are also present at those formed during S. flexneri infections.

Novel requirements for HAP2-mediated gamete fusion in Tetrahymena.

The ancestral gamete fusion protein, HAP2, catalyzes sperm-egg fusion in a broad range of taxa dating to the last eukaryotic common ancestor. Remarkably, HAP2 orthologs are structurally related to the class II fusogens of modern-day viruses, and recent studies make clear that these proteins utilize similar mechanisms to achieve membrane merger. To identify factors that may regulate HAP2 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic Δhap2 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2, whose products are necessary for the formation of membrane pores during fertilization and show that the product of a third gene, namely ZFR1, may be involved in pore maintenance and/or expansion. Finally, we propose a model that explains cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in T. thermophila's multiple mating type system.

Organelle Membrane Extensions in Mammalian Cells.

Organelles within eukaryotic cells are not isolated static compartments, instead being morphologically diverse and highly dynamic in order to respond to cellular needs and carry out their diverse and cooperative functions. One phenomenon exemplifying this plasticity, and increasingly gaining attention, is the extension and retraction of thin tubules from organelle membranes. While these protrusions have been observed in morphological studies for decades, their formation, properties and functions are only beginning to be understood. In this review, we provide an overview of what is known and still to be discovered about organelle membrane protrusions in mammalian cells, focusing on the best-characterised examples of these membrane extensions arising from peroxisomes (ubiquitous organelles involved in lipid metabolism and reactive oxygen species homeostasis) and mitochondria. We summarise the current knowledge on the diversity of peroxisomal/mitochondrial membrane extensions, as well as the molecular mechanisms by which they extend and retract, necessitating dynamic membrane remodelling, pulling forces and lipid flow. We also propose broad cellular functions for these membrane extensions in inter-organelle communication, organelle biogenesis, metabolism and protection, and finally present a mathematical model that suggests that extending protrusions is the most efficient way for an organelle to explore its surroundings.

ERK3 and DGKζ interact to modulate cell motility in lung cancer cells.

Extracellular signal-regulated kinase 3 (ERK3) promotes cell migration and tumor metastasis in multiple cancer types, including lung cancer. The extracellular-regulated kinase 3 protein has a unique structure. In addition to the N-terminal kinase domain, ERK3 includes a central conserved in extracellular-regulated kinase 3 and ERK4 (C34) domain and an extended C-terminus. However, relatively little is known regarding the role(s) of the C34 domain. A yeast two-hybrid assay using extracellular-regulated kinase 3 as bait identified diacylglycerol kinase ζ (DGKζ) as a binding partner. DGKζ was shown to promote migration and invasion in some cancer cell types, but its role in lung cancer cells is yet to be described. The interaction of extracellular-regulated kinase 3 and DGKζ was confirmed by co-immunoprecipitation and in vitro binding assays, consistent with their co-localization at the periphery of lung cancer cells. The C34 domain of ERK3 was sufficient for binding to DGKζ, while extracellular-regulated kinase 3 bound to the N-terminal and C1 domains of DGKζ. Surprisingly, in contrast to extracellular-regulated kinase 3, DGKζ suppresses lung cancer cell migration, suggesting DGKζ might inhibit ERK3-mediated cell motility. Indeed, co-overexpression of exogenous DGKζ and extracellular-regulated kinase 3 completely blocked the ability of ERK3 to promote cell migration, but DGKζ did not affect the migration of cells with stable ERK3 knockdown. Furthermore, DGKζ had little effect on cell migration induced by overexpression of an ERK3 mutant missing the C34 domain, suggesting DGKζ requires this domain to prevent ERK3-mediated increase in cell migration. In summary, this study has identified DGKζ as a new binding partner and negative regulator of extracellular-regulated kinase 3 in controlling lung cancer cell migration.

Unveiling the mono-rhamnolipid and di-rhamnolipid mechanisms of action upon plasma membrane models.

Rhamnolipids (RLs) are biosurfactants with significant tensioactive and emulsifying properties. They are mainly composed by mono-RL and di-RL components. Although there are numerous studies concerning their molecular properties, information is scarce regarding the mechanisms by which each of the two components interacts with cell membranes. Herein, we performed phase-contrast and fluorescence microscopy experiments on plasma membrane models represented by giant-unilamellar-vesicles (GUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-[[(E,2S,3R)-1,3-dihydroxy-2-(octadecanoylamino) octadec-4-enyl]peroxy-hydroxyphosphoryl]oxyethyl-trimethylazanium (sphingomyelin, SM) and (3ß)-cholest-5-en-3-ol (cholesterol, CHOL) (1:1:1 M ratio), which present liquid-order (Lo) liquid-disorder (Ld) phase coexistence, in the presence of either mono-RL or di-RL in 0.06-0.25 mM concentration range. A new method has been developed to determine area and volume of GUVs with asymmetrical shape and a kinetic model describing GUV-RL interaction in terms of two mechanisms, RL-insertion and pore formation, has been worked out. Results show that the insertion of mono-RL in the membrane outer leaflet is the dominant process with no pore formation and a negligible effect in modifying membrane permeability, but induces lipid mixing. Conversely, the di-RL-GUV interaction begins with the insertion mechanism and, as the time passes by, the pore formation process occurs. The analyses of di-RL show that the whole process is only relevant in the Ld phase with a higher extent to 0.25 mM than to 0.06 mM.

Decoding Single Cell Morphology in Osteotropic Breast Cancer Cells for Dissecting Their Migratory, Molecular and Biophysical Heterogeneity.

Breast cancer is a heterogeneous disease and the mechanistic framework for differential osteotropism among intrinsic breast cancer subtypes is unknown. Hypothesizing that cell morphology could be an integrated readout for the functional state of a cancer cell, we established a catalogue of the migratory, molecular and biophysical traits of MDA-MB-231 breast cancer cells, compared it with two enhanced bone-seeking derivative cell lines and integrated these findings with single cell morphology profiles. Such knowledge could be essential for predicting metastatic capacities in breast cancer. High-resolution microscopy revealed a heterogeneous and specific spectrum of single cell morphologies in bone-seeking cells, which correlated with differential migration and stiffness. While parental MDA-MB-231 cells showed long and dynamic membrane protrusions and were enriched in motile cells with continuous and mesenchymal cell migration, bone-seeking cells appeared with discontinuous mesenchymal or amoeboid-like migration. Although non-responsive to CXCL12, bone-seeking cells responded to epidermal growth factor with a morphotype shift and differential expression of genes controlling cell shape and directional migration. Hence, single cell morphology encodes the molecular, migratory and biophysical architecture of breast cancer cells and is specifically altered among osteotropic phenotypes. Quantitative morpho-profiling could aid in dissecting breast cancer heterogeneity and in refining clinically relevant intrinsic breast cancer subtypes.

Collective Polarization of Cancer Cells at the Monolayer Boundary.

Cell polarization, a process depending on both intracellular and intercellular interactions, is crucial for collective cell migration that commonly emerges in embryonic development, tissue morphogenesis, wound healing and cancer metastasis. Although invasive cancer cells display weak cell-cell interactions, they can invade host tissues through a collective mode. Yet, how cancer cells without stable cell-cell junctions polarize collectively to migrate and invade is not fully understood. Here, using a wound-healing assay, we elucidate the polarization of carcinoma cells at the population level. We show that with loose intercellular connections, the highly polarized leader cells can induce the polarization of following cancer cells and subsequent transmission of polarity information by membrane protrusions, leading to gradient polarization at the monolayer boundary. Unlike the polarization of epithelial monolayer where Rac1/Cdc42 pathway functions primarily, our data show that collective polarization of carcinoma cells is predominantly controlled by Golgi apparatus, a disruption of which results in the destruction of collective polarization over a large scale. We reveal that the Golgi apparatus can sustain membrane protrusion formation, polarized secretion, intracellular trafficking, and F-actin polarization, which contribute to collective cancer cell polarization and its transmission between cells. These findings could advance our understanding of collective cancer invasion in tumors.

Listeria monocytogenes Exploits Host Caveolin for Cell-to-Cell Spreading.

Listeria monocytogenes moves from one cell to another using actin-rich membrane protrusions that propel the bacterium toward neighboring cells. Despite cholesterol being required for this transfer process, the precise host internalization mechanism remains elusive. Here, we show that caveolin endocytosis is key to this event as bacterial cell-to-cell transfer is severely impaired when cells are depleted of caveolin-1. Only a subset of additional caveolar components (cavin-2 and EHD2) are present at sites of bacterial transfer, and although clathrin and the clathrin-associated proteins Eps15 and AP2 are absent from the bacterial invaginations, efficient L. monocytogenes spreading requires the clathrin-interacting protein epsin-1. We also directly demonstrated that isolated L. monocytogenes membrane protrusions can trigger the recruitment of caveolar proteins in a neighboring cell. The engulfment of these bacterial and cytoskeletal structures through a caveolin-based mechanism demonstrates that the classical nanometer-scale theoretical size limit for this internalization pathway is exceeded by these bacterial pathogens.IMPORTANCEListeria monocytogenes moves from one cell to another as it disseminates within tissues. This bacterial transfer process depends on the host actin cytoskeleton as the bacterium forms motile actin-rich membranous protrusions that propel the bacteria into neighboring cells, thus forming corresponding membrane invaginations. Here, we examine these membrane invaginations and demonstrate that caveolin-1-based endocytosis is crucial for efficient bacterial cell-to-cell spreading. We show that only a subset of caveolin-associated proteins (cavin-2 and EHD2) are involved in this process. Despite the absence of clathrin at the invaginations, the classical clathrin-associated protein epsin-1 is also required for efficient bacterial spreading. Using isolated L. monocytogenes protrusions added onto naive host cells, we demonstrate that actin-based propulsion is dispensable for caveolin-1 endocytosis as the presence of the protrusion/invagination interaction alone triggers caveolin-1 recruitment in the recipient cells. Finally, we provide a model of how this caveolin-1-based internalization event can exceed the theoretical size limit for this endocytic pathway.

Methods to Study the Roles of ß-Arrestins in Meningococcal Signaling.

Neisseria meningitidis is a Gram-negative diplococcus restricted to humans that causes severe septicemia and/or meningitidis. Initial adhesion to human endothelial cells is mediated through the interaction of type IV pili with the hetero-oligomeric complexes formed by the human receptors CD147 and the ß2-adrenergic receptor. Interaction with this complex heterodimer activates a ß-arrestin-biased signaling pathway leading to actin polymerization and accumulation of ezrin and ezrin-binding partners. These signaling events promote the formation of cell plasma membrane protrusions in endothelial cells, which are crucial for N. meningitidis colonies to resist shear stress and colonize blood vessels. Here we provide detailed protocols to evaluate the role of ß-arrestins in actin and ezrin signaling downstream of G protein-coupled receptor activation.

Retrospective comparison of perinatal outcomes following emergency cervical cerclage with or without prolapsed membranes.

OBJECTIVE: To compare perinatal outcomes following emergency cerclage between patients with singleton pregnancies with prolapsed and non-prolapsed membranes. METHODS: The present retrospective cohort study included data from women who underwent physical examination-indicated emergency cerclage at between 15 and 25 weeks of pregnancy at Saint Luc University Hospital, Brussels, Belgium, between January 1, 2000, and December 31, 2014. Outcomes were compared based on the presence of prolapsed or non-prolapsed membranes. The primary outcome measures were the duration of pregnancy at delivery and the interval between cerclage and delivery. Secondary outcomes included delivery weight, fetal or neonatal death, and neonatal morbidity, including neonatal intensive care unit admission. RESULTS: Data were included from 140 patients with cervical dilation of at least 1 cm; 85 women had non-prolapsed membranes and 55 women had prolapsed membranes. Among patients with non-prolapsed membranes, the mean duration of pregnancy at delivery was later (P<0.001), the latency between cerclage and delivery was longer (P<0.001), neonatal survival was higher (P=0.036), mean delivery weight was higher (P<0.001), the prevalence of preterm delivery was lower (P<0.001), and severe neonatal morbidity and neonatal intensive care unit admission were lower (P<0.001). CONCLUSION: Having non-prolapsed membranes was associated with improved perinatal outcomes following emergency cerclage.