Cytoskeletal motors of the dynein, kinesin and myosin superfamilies maintain and adapt subcellular organelle organization to meet functional demands and support the vesicular transport of material between organelles. These motors require the capacity to specifically recognize the vesicle/organelle to be transported and are capable of selective recognition of multiple cargo. Recent studies have begun to uncover the molecular basis for motor recruitment and have highlighted the role of organelle-associated 'cargo-adaptor' proteins in cellular transport. These adaptors possess sequences and/or structural features that enable both motor recruitment and activation from regulated, inactive, states to enable motility on the cytoskeleton. Motor-cargo adaptor interactions define a key organelle-cytoskeleton interface, acting as crucial regulatory hubs to enable the cell to finely control membrane trafficking and organelle dynamics. Understanding the molecular basis of these interactions may offer new opportunities to control and manipulate cytoskeletal and organelle dynamics for the development of new research tools and potentially therapeutics.
Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the -helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative-stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1 in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain-light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drives kinesin-1 activation.
Aim
To evaluate the in vitro immunogenic and immunomodulatory properties of induced pluripotent stem cells (iPSCs) compared with bone marrow-derived mesenchymal stromal cells (MSCs).
Materials & methods
Mouse embryonic fibroblasts (MEFs) were isolated from C3HeB/FeJ and C57BL/6J mice, and reprogrammed to generate iPSCs. Mixed leukocyte reactions were performed using MHC-matched and -mismatched responder leukocytes and stimulator leukocytes, iPSCs or MSCs. To assess immunogenic potential, iPSCs and MSCs were used as stimulator cells for responder leukocytes. To assess immunomodulatory properties, iPSCs and MSCs were cultured in the presence of stimulator and responder leukocytes. MEFs were used as a control.
Results
iPSCs had similar immunogenic properties but more potent immunomodulatory effects than MSCs. Co-culture of MHC-mismatched leukocytes with MHC-matched iPSCs resulted in significantly less responder T-cell proliferation than observed for MHC-mismatched leukocytes alone and at more responder leukocyte concentrations than with MSCs. In addition, MHC-mismatched iPSCs significantly reduced responder T-cell proliferation when co-cultured with MHC-mismatched leukocytes, while MHC-mismatched MSCs did not.
Conclusion
These results provide important information when considering the use of iPSCs in place of MSCs in both regenerative and transplantation medicine.
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