Propagating Waves of Directionality and Coordination Orchestrate Collective Cell Migration

The ability of cells to coordinately migrate in groups is crucial to enable them to travel long distances during embryonic development, wound healing and tumorigenesis, but the fundamental mechanisms underlying intercellular coordination during collective cell migration remain elusive despite considerable research efforts. A novel analytical framework is introduced here to explicitly detect and quantify cell clusters that move coordinately in a monolayer. The analysis combines and associates vast amount of spatiotemporal data across multiple experiments into transparent quantitative measures to report the emergence of new modes of organized behavior during collective migration of tumor and epithelial cells in wound healing assays. First, we discovered the emergence of a wave of coordinated migration propagating backward from the wound front, which reflects formation of clusters of coordinately migrating cells that are generated further away from the wound edge and disintegrate close to the advancing front. This wave emerges in both normal and tumor cells, and is amplified by Met activation with hepatocyte growth factor/scatter factor. Second, Met activation was found to induce coinciding waves of cellular acceleration and stretching, which in turn trigger the emergence of a backward propagating wave of directional migration with about an hour phase lag. Assessments of the relations between the waves revealed that amplified coordinated migration is associated with the emergence of directional migration. Taken together, our data and simplified modeling-based assessments suggest that increased velocity leads to enhanced coordination: higher motility arises due to acceleration and stretching that seems to increase directionality by temporarily diminishing the velocity components orthogonal to the direction defined by the monolayer geometry. Spatial and temporal accumulation of directionality thus defines coordination. The findings offer new insight and suggest a basic cellular mechanism for long-term cell guidance and intercellular communication during collective cell migration.     

Comparable Long-Term Efficacy of Lopinavir/Ritonavir and Similar Drug-Resistance Profiles in Different HIV-1 Subtypes

Background

Analysis of potentially different impact of Lopinavir/Ritonavir (LPV/r) on non-B subtypes is confounded by dissimilarities in the conditions existing in different countries. We retrospectively compared its impact on populations infected with subtypes B and C in Israel, where patients infected with different subtypes receive the same treatment.

Methods

Clinical and demographic data were reported by physicians. Resistance was tested after treatment failure. Statistical analyses were conducted using SPSS.

Results

607 LPV/r treated patients (365 male) were included. 139 had HIV subtype B, 391 C, and 77 other subtypes. At study end 429 (71%) were receiving LPV/r. No significant differences in PI treatment history and in median viral-load (VL) at treatment initiation and termination existed between subtypes. MSM discontinued LPV/r more often than others even when the virologic outcome was good (p = 0.001). VL was below detection level in 81% of patients for whom LPV/r was first PI and in 67% when it was second (P = 0.001). Median VL decrease from baseline was 1.9±0.1 logs and was not significantly associated with subtype. Median CD4 increase was: 162 and 92cells/µl, respectively, for patients receiving LPV/r as first and second PI (P = 0.001), and 175 and 98, respectively, for subtypes B and C (P<0.001). Only 52 (22%) of 237 patients genotyped while under LPV/r were fully resistant to the drug; 12(5%) were partially resistant. In48%, population sequencing did not reveal resistance to any drug notwithstanding the virologic failure. No difference was found in the rates of resistance development between B and C (p = 0.16).

Conclusions

Treatment with LPV/r appeared efficient and tolerable in both subtypes, B and C, but CD4 recovery was significantly better in virologically suppressed subtype-B patients. In both subtypes, LPV/r was more beneficial when given as first PI. Mostly, reasons other than resistance development caused discontinuation of treatment.     

Heparanase Interacts with Resistin and Augments Its Activity

In an attempt to isolate a heparanase receptor, postulated to mediate non-enzymatic functions of the heparanase protein, we utilized human urine collected from healthy volunteers. Affinity chromatography of this rich protein source on immobilized heparanase revealed resistin as a heparanase binding protein. Co-immunoprecipitation and ELISA further confirmed the interaction between heparanase and resistin. Importantly, we found that heparanase potentiates the bioactivity of resistin in its standard bioassay in which monocytic human leukemia cell line, THP1, differentiates into adherent macrophage-like foam cells. It is thus conceivable that this newly identified complex of heparanase and resistin exerts a stimulatory effect also in various inflammatory conditions known to be affected by the two proteins.     

Identification and characterization of heparin/heparan sulfate binding domains of the endoglycosidase heparanase

The endo-beta-glucuronidase, heparanase, is an enzyme that cleaves heparan sulfate at specific intra-chain sites, yielding heparan sulfate fragments with appreciable size and biological activities. Heparanase activity has been traditionally correlated with cell invasion associated with cancer metastasis, angiogenesis, and inflammation. In addition, heparanase up-regulation has been documented in a variety of primary human tumors, correlating with increased vascular density and poor postoperative survival, suggesting that heparanase may be considered as a target for anticancer drugs. In an attempt to identify the protein motif that would serve as a target for the development of heparanase inhibitors, we looked for protein domains that mediate the interaction of heparanase with its heparan sulfate substrate. We have identified three potential heparin binding domains and provided evidence that one of these is mapped at the N terminus of the 50-kDa active heparanase subunit. A peptide corresponding to this region (Lys(158)-Asp(171)) physically associates with heparin and heparan sulfate. Moreover, the peptide inhibited heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the heparan sulfate substrate. Furthermore, antibodies directed to this region inhibited heparanase activity, and a deletion construct lacking this domain exhibited no enzymatic activity. NMR titration experiments confirmed residues Lys(158)-Asn(162) as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln(270)-Lys(280)) yielded an inactive enzyme that failed to interact with cell surface heparan sulfate and hence accumulated in the culture medium of transfected HEK 293 cells to exceptionally high levels. The two heparin/heparan sulfate recognition domains are potentially attractive targets for the development of heparanase inhibitors.     

Semaphorins as Mediators of Neuronal Apoptosis

Shrinkage and collapse of the neuritic network are often observed during the process of neuronal apoptosis. However, the molecular and biochemical basis for the axonal damage associated with neuronal cell death is still unclear. We present evidence for the involvement of axon guidance molecules with repulsive cues in neuronal cell death. Using the differential display approach, an up-regulation of collapsin response mediator protein was detected in sympathetic neurons undergoing dopamine-induced apoptosis. A synchronized induction of mRNA of the secreted collapsin-1 and the intracellular collapsin response mediator protein that preceded commitment of neurons to apoptosis was detected. Antibodies directed against a conserved collapsin-derived peptide provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. Moreover, neuronal apoptosis was inhibited by antibodies against neuropilin-1, a putative component of the semaphorin III/collapsin-1 receptor. Induction of neuronal apoptosis was also caused by exposure of neurons to semaphorin III-alkaline phosphatase secreted from 293EBNA cells. Anti-collapsin-1 antibodies were effective in blocking the semaphorin III-induced death process. We therefore suggest that, before their death, apoptosis-destined neurons may produce and secrete destructive axon guidance molecules that can affect their neighboring cells and thus transfer a "death signal" across specific and susceptible neuronal populations.     

Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database

HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anti-coagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics.med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multiple matches of peptide sequences yielded 9504 IPI proteins identified with one or more peptides and 3020 proteins identified with two or more peptides (the Core Dataset). These proteins have been characterized with Gene Ontology, InterPro, Novartis Atlas, OMIM, and immunoassay-based concentration determinations. The database permits examination of many other subsets, such as 1274 proteins identified with three or more peptides. Reverse protein to DNA matching identified proteins for 118 previously unidentified ORFs. We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches. This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan. These PPP results on complexity, dynamic range, incomplete sampling, false-positive matches, and integration of diverse datasets for plasma and serum proteins lay a foundation for development and validation of circulating protein biomarkers in health and disease.     

Elements within the beta-lactoglobulin gene inhibit expression of human serum albumin cDNA and minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice.

Two new beta-lactoglobulin (BLG)/human serum albumin (HSA) hybrid gene vectors were constructed and tested for expression in COS-7 cells and in transgenic mice. The HSA sequences were inserted between the second and sixth BLG exons. Transient transfection experiments with these vectors as well as a series of additional vectors with either the BLG 5'- or 3'- intragenic sequences revealed that sequences within BLG exon 1/intron 1/exon 2 abrogated BLG- directed HSA expression in vitro, regardless of the presence of HSA introns or the origin of the 3' polyadenylation signal. In contrast, the same BLG expression cassette enabled the efficient expression of HSA cDNA or minigene in the mammary gland of transgenic mice with subsequent secretion of the corresponding protein into the milk of 56 and 82%, respectively of the mouse strains at levels up to 0.3 mg/ml. Previous attempts to express HSA cDNA inserted into exon 1 of the BLG gene had failed [Shani,M., Barash,I., Nathan,M., Ricca,G., Searfoss,G.H., Dekel,I., Faerman,A., Givol,D. and Hurwitz,D.R. (1992) Transgenic Res. 1, 195- 208]. The new BLG expression cassette conferred more stringent tissue specific expression than previously described BLG/HSA constructs [Barash,I, Faerman,A., Ratovitsky,T, Puzis,R., Nathan,M., Hurwitz,D.R. and Shani, M. (1994) Transgenic Res. 3, 141-151]. However, it was not able to insulate the transgenes from the surrounding host DNA sequences and did not result in copy number dependent expression in transgenics. Together, the in vitro and in vivo results suggest both positive and negative regulatory elements within the BLG intragenic sequences evaluated. The new BLG construct represents an extremely valuable vector for the efficient expression of cDNAs in the mammary gland of transgenic animals.     

Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy

Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.