SummaryIn vertebrates, two condensin complexes exist, condensin I and condensin II, which have differing but unresolved roles in organizing mitotic chromosomes. To dissect accurately the role of each complex in mitosis, we have made and studied the first vertebrate conditional knockouts of the genes encoding condensin I subunit CAP-H and condensin II subunit CAP-D3 in chicken DT40 cells. Live-cell imaging reveals highly distinct segregation defects. CAP-D3 (condensin II) knockout results in masses of chromatincontaining anaphase bridges. CAP-H (condensin I)-knockout anaphases have a more subtle defect, with chromatids showing fine chromatin fibres that are associated with failure of cytokinesis and cell death. Super-resolution microscopy reveals that condensin-Idepleted mitotic chromosomes are wider and shorter, with a diffuse chromosome scaffold, whereas condensin-II-depleted chromosomes retain a more defined scaffold, with chromosomes more stretched and seemingly lacking in axial rigidity. We conclude that condensin II is required primarily to provide rigidity by establishing an initial chromosome axis around which condensin I can arrange loops of chromatin.

Previous studies on the role of the tetraspanin CD37 in cellular immunity appear contradictory. In vitro approaches indicate a negative regulatory role, whereas in vivo studies suggest that CD37 is necessary for optimal cellular responses. To resolve this discrepancy, we studied the adaptive cellular immune responses of CD37 −/− mice to intradermal challenge with either tumors or model antigens and found that CD37 is essential for optimal cell-mediated immunity. We provide evidence that an increased susceptibility to tumors observed in CD37 −/− mice coincides with a striking failure to induce antigenspecific IFN-γ-secreting T cells. We also show that CD37 ablation impairs several aspects of DC function including: in vivo migration from skin to draining lymph nodes; chemotactic migration; integrin-mediated adhesion under flow; the ability to spread and form actin protrusions and in vivo priming of adoptively transferred naïve T cells. In addition, multiphoton microscopy-based assessment of dermal DC migration demonstrated a reduced rate of migration and increased randomness of DC migration in CD37 −/− mice.Together, these studies are consistent with a model in which the cellular defect that underlies poor cellular immune induction in CD37 −/− mice is impaired DC migration. Keywords: CD37 r Cellular immunity r Dendritic cells r Migration r TetraspaninsAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionAdaptive cellular immunity is initiated by presentation of foreign antigen by DCs to antigen-specific naïve T lymphocytes. DCs exist sparsely in peripheral tissues in a state specialized for antigen uptake and processing. However, upon pathogen encounter, DCsCorrespondence: Dr. Mark D. Wright e-mail: Mark.Wright@monash.edu transduce signals through pattern recognition receptors, leading to an increased expression of cell surface molecules and cytokines, and induction of DC migration from the periphery to draining lymph nodes (DLNs) via afferent lymphatic vessels. Thus, upon their arrival in secondary lymphoid organs, DCs are equipped to initiate adaptive cellular immune responses through their ability to * These authors contributed equally to this work.C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2013. 43: 1208-1219 Cellular immune response 1209 activate naïve antigen-specific T cells [1]. Despite the importance of DC migration from the periphery to DLNs, the roles of the numerous molecules that regulate this process are incompletely understood. One such molecule is the leukocyte-specific membrane protein CD37, a member of the tetraspanin protein superfamily. Tetraspanins molecularly organize cellular membranes by interactions with partner molecules, which they direct into regulated signal-transducing tetraspanin-enriched microdomains. The cellular processes regulated by tetraspanin-mediated molecular organization include proliferation, adhesion and migration [2,3]. In immune cells, many important cell s...

BackgroundCell biologists face the need to rapidly analyse their proteins of interest in order to gain insight into their function. Often protein purification, cellular localisation and Western blot analyses can be multi-step processes, where protein is lost, activity is destroyed or effective antibodies have not yet been generated.AimTo develop a method that simplifies the critical protein analytical steps of the laboratory researcher, leading to easy, efficient and rapid protein purification, cellular localisation and quantification.ResultsWe have tagged the SMC2 subunit of the condensin complex with the Streptavidin-Binding Peptide (SBP), optimising and demonstrating the efficacious use of this tag for performing these protein analytical steps. Based on silver staining, and Western analysis, SBP delivered an outstanding specificity and purity of the condensin complex. We also developed a rapid and highly specific procedure to localise SBP-tagged proteins in cells in a single step procedure thus bypassing the need for using antibodies. Furthermore we have shown that the SBP tag can be used for isolating tagged proteins from chemically cross-linked cell populations for capturing DNA-protein interactions.ConclusionsThe small 38-amino acid synthetic SBP offers the potential to successfully perform all four critical analytical procedures as a single step and should have a general utility for the study of many proteins and protein complexes.