Stem Cell Marker Expression in Early Stage Colorectal Cancer is Associated with Recurrent Intestinal Neoplasia.

BACKGROUND: Colorectal cancer (CRC) ranks second in cancer deaths worldwide and presents multiple management challenges, one of which is identifying high risk stage II disease that may benefit from adjuvant therapy. Molecular biomarkers, such as ones that identify stem cell activity, could better stratify high-risk cohorts for additional treatment. METHODS: To identify possible biomarkers of high-risk disease in early-stage CRC, a discovery set (n = 66) of advanced-stage tumors were immunostained with antibodies to stemness proteins (CD166, CD44, CD26, and LGR5) and then digitally analyzed. Using a second validation cohort (n = 54) of primary CRC tumors, we analyzed protein and gene expression of CD166 across disease stages, and extended our analyses to CD166-associated genes (LGR5, ASCL2, BMI1, POSTN, and VIM) by qRT-PCR. RESULTS: Stage III and metastatic CRC tumors highly expressed stem cell-associated proteins, CD166, CD44, and LGR5. When evaluated across stages, CD166 protein expression was elevated in advanced-stage compared to early-stage tumors. Notably, a small subset of stage I and II cancers harbored elevated CD166 protein expression, which correlated with development of recurrent cancer or adenomatous polyps. Gene expression analyses of CD166-associated molecules revealed elevated ASCL2 in primary tumors from patients who recurred. CONCLUSIONS: We identified a protein signature prognostic of aggressive disease in early stage CRC. Stem cell-associated protein and gene expression identified a subset of early-stage tumors associated with cancer recurrence and/or subsequent adenoma formation. Signatures for stemness offer promising fingerprints for stratifying early-stage patients at high risk of recurrence.

Chemorepellent Semaphorin 3E Negatively Regulates Neutrophil Migration In Vitro and In Vivo.

Neutrophil migration is an essential step in leukocyte trafficking during inflammatory responses. Semaphorins, originally discovered as axon guidance cues in neural development, have been shown to regulate cell migration beyond the nervous system. However, the potential contribution of semaphorins in the regulation of neutrophil migration is not well understood. This study examines the possible role of a secreted chemorepellent, Semaphorin 3E (Sema3E), in neutrophil migration. In this study, we demonstrated that human neutrophils constitutively express Sema3E high-affinity receptor, PlexinD1. Sema3E displayed a potent ability to inhibit CXCL8/IL-8-induced neutrophil migration as determined using a microfluidic device coupled to real-time microscopy and a transwell system in vitro. The antimigratory effect of Sema3E on human neutrophil migration was associated with suppression of CXCL8/IL-8-mediated Ras-related C3 botulinum toxin substrate 1 GTPase activity and actin polymerization. We further addressed the regulatory role of Sema3E in the regulation of neutrophil migration in vivo. Allergen airway exposure induced higher neutrophil recruitment into the lungs of Sema3e-/- mice compared with wild-type controls. Administration of exogenous recombinant Sema3E markedly reduced allergen-induced neutrophil recruitment into the lungs, which was associated with alleviation of allergic airway inflammation and improvement of lung function. Our data suggest that Sema3E could be considered an essential regulatory mediator involved in modulation of neutrophil migration throughout the course of neutrophilic inflammation.

Reelin-Dab1 signaling system in human colorectal cancer.

Reelin is an extracellular matrix protein that plays a critical role in neuronal migration. Here we show that the mucosa of human colon expresses reelin, its receptors ApoER2 and VLDLR, and its effector protein Dab1. Immunohistochemical analyses reveal that reelin expression is restricted to pericryptal myofibroblasts; Dab1 is detected at myofibroblasts, the apical domain of surface epithelial and crypt cells, and a strong linear staining is observed at the basement membrane; VLDLR and ApoER2 are in the cytoplasm of surface epithelium and myofibroblasts, and VLDLR is also detected in the cytoplasm of the crypt cells. Human colorectal cancer downregulates reelin without change in vimentin or N-cadherin mRNA levels. Decreased Reelin mRNA expression is accompanied by decreased HIC1 mRNA levels, increased mRNA levels of ApoER2 and DNMT1, increased reelin hypermethylation and no change in either Cask or TGF-ß1 mRNAs, suggesting that reelin repression results from a DNMT1-mediated hypermethylation of the reelin gene promoter. Decreased HIC1 expression may repress reelin transcription via increasing ApoER2 transcription. We conclude that the mucosa of human colon expresses the reelin-Dab1 signaling system and that reelin is repressed in colorectal cancer before epithelial-mesenchymal transition has occurred. The significant down-regulation of reelin expression makes this gene a promising biomarker for colorectal cancers. © 2016 Wiley Periodicals, Inc.

Effects of conventional CPB and mini-CPB on neutrophils CD162, CD166 and CD195 expression.

OBJECTIVE: Cardiac surgery is known to trigger a systemic inflammatory response. While the use of conventional cardiopulmonary bypass (CPB) results in profound inflammation, modified mini-CPB is considered less harmful. We evaluated the impact of cardiac surgery on the expression of CD162, CD166, CD195 molecules and their association with the type of CPB used. METHODS AND RESULTS: Twenty-four patients were enrolled in our study. Twelve of them were operated using conventional CPB while the other twelve patients underwent surgery with mini-CPB. Blood samples were analysed by flow cytometry. We observed a significant increase in median fluorescence intensity of CD162 and CD195 that peaked instantly after surgery and normalized to the baseline value on the 1st day post surgery, whereas CD166 was initially down-regulated and its median fluorescence intensity (MFI) value increased to the baseline in the next few days. CONCLUSION: We observed immediate changes in the expression of CD162, CD166, and CD195 molecules on the neutrophils after surgery in both study groups of patients. The intensity of the observed changes was significantly greater in the group of patients who underwent conventional CPB compared to patients who underwent mini-CPB cardiac surgery.

Progenitor Cells from Cartilage: Grade Specific Differences in Stem Cell Marker Expression.

Recent research has confirmed the presence of Mesenchymal stem cell (MSC)-like progenitors (MPC) in both normal and osteoarthritic cartilage. However, there is only limited information concerning how MPC markers are expressed with osteoarthritis (OA) progression. The purpose of this study was to compare the prevalence of various MPC markers in different OA grades. Human osteoarthritic tibial plateaus were obtained from ten patients undergoing total knee replacement. Each sample had been classified into a mild or severe group according to OARSI scoring. Tissue was taken from each specimen and mRNA expression levels of CD105, CD166, Notch 1, Sox9, Acan and Col II A1 were measured at day 0 and day 14 (2 weeks in vitro). Furthermore, MSC markers: Nucleostemin, CD90, CD73, CD166, CD105 and Notch 1 were studied by immunofluorescence. mRNA levels of MSC markers did not differ between mild and severe OA at day 0. At day 14, protein analysis showed that proliferated cells from both sources expressed all 6 MSC markers. Only cells from the mild OA subjects resulted in a significant increase of mRNA CD105 and CD166 after in vitro expansion. Moreover, cells from the mild OA subjects showed significantly higher levels of CD105, Sox9 and Acan compared with those from severe OA specimens. Results confirmed the presence of MSC markers in mild and severe OA tissue at both mRNA and protein levels. We found significant differences between cells obtained from mild compared to severe OA specimens suggests that mild OA derived cells may have a greater MSC potential.

Prox1 Regulates the Subtype-Specific Development of Caudal Ganglionic Eminence-Derived GABAergic Cortical Interneurons.

Neurogliaform (RELN+) and bipolar (VIP+) GABAergic interneurons of the mammalian cerebral cortex provide critical inhibition locally within the superficial layers. While these subtypes are known to originate from the embryonic caudal ganglionic eminence (CGE), the specific genetic programs that direct their positioning, maturation, and integration into the cortical network have not been elucidated. Here, we report that in mice expression of the transcription factor Prox1 is selectively maintained in postmitotic CGE-derived cortical interneuron precursors and that loss of Prox1 impairs the integration of these cells into superficial layers. Moreover, Prox1 differentially regulates the postnatal maturation of each specific subtype originating from the CGE (RELN, Calb2/VIP, and VIP). Interestingly, Prox1 promotes the maturation of CGE-derived interneuron subtypes through intrinsic differentiation programs that operate in tandem with extrinsically driven neuronal activity-dependent pathways. Thus Prox1 represents the first identified transcription factor specifically required for the embryonic and postnatal acquisition of CGE-derived cortical interneuron properties. SIGNIFICANCE STATEMENT: Despite the recognition that 30% of GABAergic cortical interneurons originate from the caudal ganglionic eminence (CGE), to date, a specific transcriptional program that selectively regulates the development of these populations has not yet been identified. Moreover, while CGE-derived interneurons display unique patterns of tangential and radial migration and preferentially populate the superficial layers of the cortex, identification of a molecular program that controls these events is lacking.Here, we demonstrate that the homeodomain transcription factor Prox1 is expressed in postmitotic CGE-derived cortical interneuron precursors and is maintained into adulthood. We found that Prox1 function is differentially required during both embryonic and postnatal stages of development to direct the migration, differentiation, circuit integration, and maintenance programs within distinct subtypes of CGE-derived interneurons.

The Neurexin/N-Ethylmaleimide-sensitive Factor (NSF) Interaction Regulates Short Term Synaptic Depression.

Although Neurexins, which are cell adhesion molecules localized predominantly to the presynaptic terminals, are known to regulate synapse formation and synaptic transmission, their roles in the regulation of synaptic vesicle release during repetitive nerve stimulation are unknown. Here, we show that nrx mutant synapses exhibit rapid short term synaptic depression upon tetanic nerve stimulation. Moreover, we demonstrate that the intracellular region of NRX is essential for synaptic vesicle release upon tetanic nerve stimulation. Using a yeast two-hybrid screen, we find that the intracellular region of NRX interacts with N-ethylmaleimide-sensitive factor (NSF), an enzyme that mediates soluble NSF attachment protein receptor (SNARE) complex disassembly and plays an important role in synaptic vesicle release. We further map the binding sites of each molecule and demonstrate that the NRX/NSF interaction is critical for both the distribution of NSF at the presynaptic terminals and SNARE complex disassembly. Our results reveal a previously unknown role of NRX in the regulation of short term synaptic depression upon tetanic nerve stimulation and provide new mechanistic insights into the role of NRX in synaptic vesicle release.

Isolation and characterization of human mesenchymal stem cells from gingival connective tissue.

BACKGROUND AND OBJECTIVE: The main purpose of this study was to isolate and characterize gingival connective tissue-derived mesenchymal stem cells (GMSCs). The secondary purpose was to present a modified isolation method for the GMSCs. MATERIAL AND METHODS: Collected healthy gingival tissue samples were de-epithelialized and minced into small fragments. The tissues were digested by dispase and collagenase IV for 30 min. The first digested cell suspension was discarded, and then additional digestion was performed to the remaining cells in the same solution for 90 min. The isolated cells from gingiva was incubated in 37°C humidified condition and observed by inverted microscope. Cytoskeletal morphology was evaluated by phalloidin immunofluorescence. Potency of the cells was tested by colony-forming unit fibroblast assay. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometric, immunofluorescence analysis. RESULTS: GMSCs showed spindle-shaped, fibroblast-like morphology, colony-forming abilities, adherence to plastic and multilineage differentiation (osteogenic, adipogenic, chondrogenic) potency. GMSCs expressed CD44, CD73, CD90 and CD105, but did not express CD14, CD45, CD34 and CD19 in flow cytometry. Expression of stem cell markers (SSEA-4, STRO-1, CD146, CD166 and CD271) and a mesenchymal marker (vimentin) were observed by immunofluorescence. CONCLUSIONS: In conclusion, we isolated and characterized stem cells from human gingival connective tissue with modified protocol. GMSCs showed multipotency with high proliferation and characteristics of mesenchymal stem cells. GMSCs are promising sources for tissue engineering and may be obtained during routine procedures under local anesthesia. Further research is needed to evaluate the potential of GSMCs' proliferation and cryopreservation.

Development of layer 1 neurons in the mouse neocortex.

Layer 1 of the neocortex harbors a unique group of neurons that play crucial roles in synaptic integration and information processing. Although extensive studies have characterized the properties of layer 1 neurons in the mature neocortex, it remains unclear how these neurons progressively acquire their distinct morphological, neurochemical, and physiological traits. In this study, we systematically examined the dynamic development of Cajal-Retzius cells and γ-aminobutyric acid (GABA)-ergic interneurons in layer 1 during the first 2 postnatal weeks. Cajal-Retzius cells underwent morphological degeneration after birth and gradually disappeared from layer 1. The majority of GABAergic interneurons showed clear expression of at least 1 of the 6 distinct neurochemical markers, including Reelin, GABA-A receptor subunit delta (GABAARδ), neuropeptide Y, vasoactive intestinal peptide (VIP), calretinin, and somatostatin from postnatal day 8. Furthermore, according to firing pattern, layer 1 interneurons can be divided into 2 groups: late-spiking (LS) and burst-spiking (BS) neurons. LS neurons preferentially expressed GABAARδ, whereas BS neurons preferentially expressed VIP. Interestingly, both LS and BS neurons exhibited a rapid electrophysiological and morphological development during the first postnatal week. Our results provide new insights into the molecular, morphological, and functional developments of the neurons in layer 1 of the neocortex.

Prenatal exposure to the CB1 and CB2 cannabinoid receptor agonist WIN 55,212-2 alters migration of early-born glutamatergic neurons and GABAergic interneurons in the rat cerebral cortex.

The endocannabinoid system, composed of cannabinoid receptors, endocannabinoids, and synthesis and degradation enzymes, is present since early stages of brain development. During this period, the endocannabinoid system is involved in the regulation of neural progenitor proliferation and specification as well as the migration and differentiation of pyramidal neurons and interneurons. Marijuana consumption during pregnancy represents a serious risk in relation to the fetal brain development since Δ(9) -tetrahidrocannabinol, the main active compound of cannabis, can reach the fetus through placenta and hemato-encephalic barrier. Cohort studies performed on children and adolescents of mothers who consumed marijuana during pregnancy reported cognitive and comportamental abnormalities. In the present study, we examined the expression of the cannabinoid receptor CB1 R during corticogenesis in radially and tangentially migrating post-mitotic neurons. We found that prenatal exposure to WIN impaired tangential and radial migration of post-mitotic neurons in the dorsal pallium. In addition, we described alterations of two transcription factors associated with proliferating and newly post-mitotic glutamatergic cells in the dorsal pallium, Tbr1 and Tbr2, and disruption in the number of Cajal-Retzius cells. The present results contribute to the knowledge of neurobiological substrates that determine neuro-comportamental changes that will persist through post-natal life.

A protocol for concurrent high-quality immunohistochemical and biochemical analyses in adult mouse central nervous system.

Biochemical analysis of central nervous system proteins and nucleic acids requires fresh-tissue homogenates, whereas immunohistochemistry usually is performed in sections prepared from perfusion-fixed tissue. Post-mortem immersion-fixation is possible, but largely impairs morphological preservation and protein antigenicity. Here, we present a simple, fast and versatile protocol allowing concurrent biochemical and immunohistochemical analysis, including pre-embedding immunoelectron microscopy, using tissue from the same animal. The protocol includes a brief transcardiac perfusion with ice-cold, oxygenated and glucose-supplemented artificial cerebrospinal fluid to maintain brain tissue alive, prior to isolation of regions of interest, followed by homogenisation for biochemistry or immersion-fixation for immunohistochemistry. We provide several examples demonstrating that this protocol allows optimal biochemical and morphological analysis, characterised with optimal sensitivity and preservation of tissue structure, along with a reduction of artefacts typically seen in perfusion-fixed tissue. This protocol should find widespread applications for combining analytical methods in tissue from the same animal, thereby reducing the number of mice required for a given experiment.

Imaging multiple biomarkers in captured rare cells by sequential immunostaining and photobleaching.

Several technologies recently have been developed for separating and counting circulating tumor cells (CTCs) in the human blood. CTCs play an important role in the metastasis of cancer. Most of the current applications are focused on the enumeration of CTCs; however, analysis of the enumerated CTCs has been proven to be increasingly important. Ensemble-decision aliquot ranking (eDAR) is a high-throughput method that allows the isolation of the CTCs from the whole blood with high recovery and a zero false-positive rate. Coupling a CTC separation and capturing method, such as eDAR, with a downstream immunostaining test provides information about the cell's expression of certain protein biomarkers. In this article, we introduce a semi-automated system for sequential immunolabeling and photobleaching on the eDAR platform. With our new technique, we were able to evaluate the expression of eight different biomarkers on isolated CTCs.

Transplantation of human dental pulp stem cells: enhance bone consolidation in mandibular distraction osteogenesis.

PURPOSE: The main aim of the present study was to evaluate the capacity of stem cells from human exfoliated deciduous teeth (SHED) to enhance mandibular distraction osteogenesis (DO) in rabbits. MATERIALS AND METHODS: A randomized controlled trial was conducted. Eighteen skeletally immature New Zealand white rabbits were divided into 2 groups, with 9 in the control group and 9 in the SHED group. The SHED were isolated, expanded, and characterized. Six million cells were transplanted into the distracted area during the osteotomy period. After a 4-day latency period, a total of 6 mm was distracted for 6 days. The newly formed bone was analyzed radiologically, histologically, and histomorphometrically at 2, 4, and 6 weeks postoperatively. Nonparametric analysis of variance (Kruskal-Wallis test) was used for data analysis, and P < .05 was considered statistically significant. RESULTS: The cell lineage was positive for the 2 mesenchymal stem cell markers tested (CD105 and CD166). More mature bone in the SHED transplanted group was observed radiographically and histologically. Histomorphologically, the percentage of newly formed bone after 2, 4, and 6 weeks was 18.41% and 41.53%, 31.68% and 59.78%, and 52.34% and 65.24% in the control and SHED groups, respectively. The difference between the groups was statistically significant (P = .012). The bone union and stage of bone maturity scores were significantly different between the control and SHED groups (P = .006 and P = .011, respectively). CONCLUSIONS: Our findings suggest that SHED can serve as an additional cell resource for DO enhancement in rabbits and might be a promising model for the reconstruction of large mandibular defects in human oral maxillofacial surgery.

Comparison of immuno-phenotypes of stem cells from human dental pulp and periodontal ligament.

It has been established that human dental pulp and periodontal ligament contain a population of mesenchymal stem cells (MSCs). However, the phenotypic analysis in terms of putative stem cell markers expressed by these stem cell populations is incomplete. It is relevant to understand whether stem cells derived from closely related tissues are programmed differently. The aim of the present study is to analyze whether these stem cells depict distinct characteristics by gaining insight into differences in their immunophenotype. Dental pulp and periodontal ligament tissue samples were obtained from extracted impacted wisdom teeth. Cell cultures were analyzed for surface and intracellular markers by indirect immunoflourescence. Detailed immunophenotype analysis was carried out by flow cytometry using relevant markers. The present study data shows dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) expressed embryonic stem (ES) cell markers Oct-4, Nanog and mesodermal marker Vimentin by indirect immunoflourescence. PDLSCs, however, had a weak expression of Nanog. Immunophenotyping revealed strong expression of MSC markers (CD73, CD90) in DPSCs and PDLSCs. Differences were observed in expression of stemness-related markers. DPSCs displayed increased percentages of SSEA4, CD13 and CD166 and decreased CD9 expression compared to PDLSCs. Both stem cells express common MSC markers, different levels of expression suggests there might be more than one stem cell population existing within these tissues which differ in their embryonic status, and DPSCs are a more primitive stem cell population in comparison to PDLSCs.

The analysis of neural recognition molecules: benefits and vicissitudes of functional knock-outs using antibodies and gene ablation.

Altering or removing neural recognition molecules by blocking antibodies or genetic deletion has led to a flurry of predictions concerning their function. Each experimental approach has advantages and disadvantages that have to be considered when interpreting the resulting phenotypes.

Tissue microarray analysis of ezrin, KBA.62, CD166, nestin, and p-Akt in melanoma versus banal and atypical nevi, and nonmelanocytic lesions.

Multiple melanocytic markers are useful for differentiating between melanoma and nonmelanocytic lesions but generally do not distinguish melanoma from nevi and atypical melanocytic lesions. We sought to determine if several immunohistochemical markers recently described in the literature, including ezrin, KBA.62, p-Akt, CD166, and nestin, may be helpful in distinguishing these lesions. One hundred ten tissue microarray samples were scored for nestin and CD166 and 220 samples for ezrin, KBA.62, and p-Akt. We found that putative stem cell markers nestin and CD166 were both expressed in most melanomas (86% and 65% of samples, respectively), including desmoplastic melanoma, but were also expressed at similar levels in nevi (79% and 74%, respectively). In addition, these markers were not specific for melanocytic lesions. Ezrin was also expressed in both nevi and melanoma (81% each), including desmoplastic melanoma (75%), and in neural tumors. KBA.62 stained more cases of nevi versus melanoma (93% and 65%, respectively) and was positive in 53% of desmoplastic melanoma. However, it was also positive in several nonmelanocytic tumors. P-Akt expression was generally weak but was increased in nevi (75%) versus melanoma (43%), and was lost in desmoplastic melanomas (5%). Overall, only KBA.62 and p-Akt expression differed between melanoma and nevi, and none of these markers were completely specific for melanocytic tumors versus nonmelanocytic lesions.

The septate junction protein caspr is required for structural support and retention of KCNQ4 at calyceal synapses of vestibular hair cells.

The afferent innervation contacting the type I hair cells of the vestibular sensory epithelia form distinct calyceal synapses. The apposed presynaptic and postsynaptic membranes at this large area of synaptic contact are kept at a remarkably regular distance. Here, we show by freeze-fracture electron microscopy that a patterned alignment of proteins at the calyceal membrane resembles a type of intercellular junction that is rare in vertebrates, the septate junction (SJ). We found that a core molecular component of SJs, Caspr, colocalizes with the K(+) channel KCNQ4 at the postsynaptic membranes of these calyceal synapses. Immunolabeling and ultrastructural analyses of Caspr knock-out mice reveal that, in the absence of Caspr, the separation between the membranes of the hair cells and the afferent neurons is conspicuously irregular and often increased by an order of magnitude. In these mutants, KCNQ4 fails to cluster at the postsynaptic membrane and appears diffused along the entire calyceal membrane. Our results indicate that a septate-like junction provides structural support to calyceal synaptic contact with the vestibular hair cell and that Caspr is required for the recruitment or retention of KCNQ4 at these synapses.

Knockout of HIF-1α in tumor-associated macrophages enhances M2 polarization and attenuates their pro-angiogenic responses.

Tumor-associated macrophages (TAMs) constitute major infiltrates of solid tumors and express a marker profile that characterizes alternatively activated macrophages (MФs). TAMs accumulate in hypoxic tumor regions, express high amounts of hypoxia-inducible factor-1 (HIF-1) and contribute to tumor angiogenesis and invasiveness. However, the precise role of HIF-1 on MФ infiltration and phenotype alterations remains poorly defined. Therefore, we cocultured wild type (wt) versus HIF-1α(-/-) MФs with tumor spheroids. Both, wt and HIF-1α(-/-) MФs, infiltrated hypoxic regions of tumor spheroids at equal rates and got alternatively activated. Interestingly, significantly higher amounts of HIF-1α(-/-) MФs expressed the TAM markers CD206 and stabilin-1 compared with wt phagocytes. Stimulation of infiltrated TAMs with lipopolysaccharide (LPS)/interferon-γ revealed a reduced expression of the pro-inflammatory markers interleukin (IL)-6, tumor necrosis factor-α and inducible nitric oxide synthase in HIF-1α(-/-) MФs. Furthermore, HIF-1α(-/-) MФs were less cytotoxic toward tumor cells. Although infiltration of MФs increased the invasive potential of tumor spheroids independently of HIF-1, the ability to stimulate differentiation of stem cells toward CD31-positive cells was triggered by wt but not by HIF-1α(-/-) MФs. Our data suggest that HIF-1α-deficient MФs develop a more prominent TAM marker profile accompanied by reduced cytotoxicity, whereas HIF-1 seems indispensable for the angiogenesis-promoting properties of TAMs.

Biophysical mechanisms underlying the membrane trafficking of synaptic adhesion molecules.

Adhesion proteins play crucial roles at synapses, not only by providing a physical trans-synaptic linkage between axonal and dendritic membranes, but also by connecting to functional elements including the pre-synaptic neurotransmitter release machinery and post-synaptic receptors. To mediate these functions, adhesion proteins must be organized on the neuronal surface in a precise and controlled manner. Recent studies have started to describe the mobility, nanoscale organization, and turnover rate of key synaptic adhesion molecules including cadherins, neurexins, neuroligins, SynCAMs, and LRRTMs, and show that some of these proteins are highly mobile in the plasma membrane while others are confined at sub-synaptic compartments, providing evidence for different regulatory pathways. In this review article, we provide a biophysical view of the diffusional trapping of adhesion molecules at synapses, involving both extracellular and intracellular protein interactions. We review the methodology underlying these measurements, including biomimetic systems with purified adhesion proteins, means to perturb protein expression or function, single molecule imaging in cultured neurons, and analytical models to interpret the data. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.