Cells sense their physical environment through mechanotransduction-that is, by translating mechanical forces and deformations into biochemical signals such as changes in intracellular calcium concentration or activation of diverse signalling pathways. In turn, these signals can adjust cellular and extracellular structure. This mechanosensitive feedback modulates cellular functions as diverse as migration, proliferation, differentiation, and apoptosis and is critical for organ development and homeostasis. Consequently, defects in mechanotransduction-often caused by mutations or misregulation of proteins that disturb cellular or extracellular mechanics-are implicated in the development of a wide array of diseases, ranging from muscular dystrophies and cardiomyopathies to cancer progression and metastasis.Mechanotransduction describes the cellular processes that translate mechanical stimuli into biochemical signals, thus allowing cells to adapt to their physical environment. Extensive research over the last decades has identified several molecular players involved in cellular mechanotransduction (Box 1); however, many components, and especially the identity of the primary mechanosensor(s), remain incompletely defined.Research in mechanotransduction has often focused on sensory cells, such as hair cells in the inner ear. These specialized cells often have evolved specific cellular structures ( Fig. 1) that are tailored to transduce mechanical inputs into biochemical signals (for example, by opening ion channels in response to applied forces) and thus provide a good model to study cellular mechanosensing. Subsequently, it has become apparent that mechanotransduction signalling has a critical role in the maintenance of many mechanically stressed tissues such as muscle, bone, cartilage, and blood vessels; consequentially, research has expanded to diverse celltypes such as myocytes, endothelial cells, and vascular smooth muscle cells. Mechanotransduction is now emerging to be involved in a much broader range of cellular functions, not just in a subset of specialized cells and tissues. For example, stem-cell differentiation can be steered towards specific fates based on the geometry and stiffness of the substrate on which the cells are grown on 1 , and intercellular physical interactions such as tension and adhesion might be as important in embryonic development as concentration gradients of morphogenic factors (see the Review by Wozniak and Chen in this issue.)In this Review, we discuss how mutations and modifications that interfere with normal mechanotransduction and cellular sensitivity to mechanical stress could be implicated in a wide spectrum of diseases that range from loss of hearing to muscular dystrophies and cancer (Table 1). Many of these diseases share few similarities at first sight. How could muscular dystrophies be related to atherosclerosis or kidney failure? In the following, we will highlight some of these disorders and discuss how they could be traced back, at least in part, to defects in mec...

Maintaining physical connections between the nucleus and the cytoskeleton is important for many cellular processes that require coordinated movement and positioning of the nucleus. Nucleo-cytoskeletal coupling is also necessary to transmit extracellular mechanical stimuli across the cytoskeleton to the nucleus, where they may initiate mechanotransduction events. The LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, formed by the interaction of nesprins and SUN proteins at the nuclear envelope, can bind to nuclear and cytoskeletal elements; however, its functional importance in transmitting intracellular forces has never been directly tested. This question is particularly relevant since recent findings have linked nesprin mutations to muscular dystrophy and dilated cardiomyopathy. Using biophysical assays to assess intracellular force transmission and associated cellular functions, we identified the LINC complex as a critical component for nucleo-cytoskeletal force transmission. Disruption of the LINC complex caused impaired propagation of intracellular forces and disturbed organization of the perinuclear actin and intermediate filament networks. Although mechanically induced activation of mechanosensitive genes was normal (suggesting that nuclear deformation is not required for mechanotransduction signaling) cells exhibited other severe functional defects after LINC complex disruption; nuclear positioning and cell polarization were impaired in migrating cells and in cells plated on micropatterned substrates, and cell migration speed and persistence time were significantly reduced. Taken together, our findings suggest that the LINC complex is critical for nucleo-cytoskeletal force transmission and that LINC complex disruption can result in defects in cellular structure and function that may contribute to the development of muscular dystrophies and cardiomyopathies.A stable connection between the nucleus and cytoskeleton is required for a wide range of physiological functions such as cell migration or nuclear positioning. Two recently discovered major molecular components involved in nucleo-cytoskeletal coupling are nesprin and SUN proteins, nuclear envelope transmembrane protein families that form a bridge across the nuclear envelope. SUN1 and SUN2 are retained at the inner nuclear membrane by their interaction with lamins, nuclear pore complex proteins, and the nuclear interior, whereas their conserved C-terminal SUN domain extends into the perinuclear space (1-3). Here, they interact with the highly conserved C-terminal KASH domain of nesprins located at the nuclear envelope. Four nesprin genes have been identified to date, many of them containing diverse isoforms as a result of alternate initiation and splicing sites. The largest isoforms of nesprins-1 and -2 contain an N-terminal actin-binding domain, enabling them to interact with cytoplasmic actin filaments (4, 5). Through spectrin-repeat-mediated interactions with kinesin and/or dynein subunits, nesprins-1 and -2 can also connect to microtubules (6...

Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss Muscular Dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome (HGPS).1 The majority of LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and disturbed interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes.1 We report here that lamin A/C-deficient (Lmna−/−) and Lmna N195K mutant cells have impaired nuclear translocation and downstream signaling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function.2 Disturbed nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna−/− and N195K mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease etiology for the cardiac phenotype in many laminopathies, whereby lamins A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization.

Background: The unusual nuclear shape of neutrophils has been speculated to facilitate their passage through confined spaces. Results: Levels of nuclear protein lamin A modulate cell passage through micron-scale pores. Conclusion:The unique protein composition of neutrophil nuclei facilitates their deformation; lobulated nuclear shape is not essential. Significance: Altered nuclear envelope composition, as reported in cancer cells, could impact cell passage through physiological gaps.

Lamins are intermediate filament proteins that assemble into a meshwork underneath the inner nuclear membrane, the nuclear lamina. Mutations in the LMNA gene, encoding lamins A and C, cause a variety of diseases collectively called laminopathies. The disease mechanism for these diverse conditions is not well understood. Since lamins A and C are fundamental determinants of nuclear structure and stability, we tested whether defects in nuclear mechanics could contribute to the disease development, especially in laminopathies affecting mechanically stressed tissue such as muscle. Using skin fibroblasts from laminopathy patients and lamin A/C-deficient mouse embryonic fibroblasts stably expressing a broad panel of laminopathic lamin A mutations, we found that several mutations associated with muscular dystrophy and dilated cardiomyopathy resulted in more deformable nuclei; in contrast, lamin mutants responsible for diseases without muscular phenotypes did not alter nuclear deformability. We confirmed our results in intact muscle tissue, demonstrating that nuclei of transgenic Drosophila melanogaster muscle expressing myopathic lamin mutations deformed more under applied strain than controls. In vivo and in vitro studies indicated that the loss of nuclear stiffness resulted from impaired assembly of mutant lamins into the nuclear lamina. Although only a subset of lamin mutations associated with muscular diseases caused increased nuclear deformability, almost all mutations tested had defects in force transmission between the nucleus and cytoskeleton. In conclusion, our results indicate that although defective nuclear stability may play a role in the development of muscle diseases, other factors, such as impaired nucleo-cytoskeletal coupling, likely contribute to the muscle phenotype.

Targeted drug delivery offers an opportunity for the development of safer and more effective therapies for the treatment of cancer. In this study, we sought to identify short, cell-internalizing peptide ligands that could serve as directive agents for specific drug delivery in hematologic malignancies. By screening of human leukemia cells with a combinatorial phage display peptide library, we isolated a peptide motif, sequence Phe-Phe/TyrAny-Leu-Arg-Ser (F F / Y XLRS), which bound to different leukemia cell lines and to patient-derived bone marrow samples. The motif was internalized through a receptor-mediated pathway, and we next identified the corresponding receptor as the transmembrane glycoprotein neuropilin-1 (NRP-1). Moreover, we observed a potent anti-leukemia cell effect when the targeting motif was synthesized in tandem to the pro-apoptotic sequence D (KLAKLAK) 2 . Finally, our results confirmed increased expression of NRP-1 in representative human leukemia and lymphoma cell lines and in a panel of bone marrow specimens obtained from patients with acute lymphoblastic leukemia or acute myelogenous leukemia compared with normal bone marrow. These results indicate that NRP-1 could potentially be used as a target for liganddirected therapy in human leukemias and lymphomas and that the prototype CGFYWLRSC-GG-D (KLAKLAK) 2 is a promising drug candidate in this setting. (Blood. 2011;117(3):920-927) IntroductionThe development of targeted drug-delivery strategies for safer and more effective therapy in human hematologic malignancies has been a long-standing goal for clinical and basic investigators. We reasoned that profiling of human leukemia-and lymphoma-derived cell lines with combinatorial libraries might yield ligand peptide sequences that bind to specific internalizing receptors on cell surfaces and may potentially lead to the discovery of new or unrecognized therapeutic targets. Such targeting motifs could also serve as vehicles for the preferential delivery of cytotoxic agents to leukemia and lymphoma cells.Several cell surface-binding peptides recognizing receptors in the membranes of lymphoma and leukemia cell lines have been reported. [1][2][3][4][5] The selected peptide ligands are readily internalized by cells and may therefore be potentially useful in ligand-directed drug delivery. Recently, we described an arginine-rich motif that is internalized into leukemia and lymphoma cells through the macropinocytotic pathway; however, the precise cell surface receptor has yet to be identified. 6 In effect, there is currently a relative lack of well-defined ligand-receptor systems for targeting human leukemia or lymphoma cells. The identification and validation of ligandreceptor pairs for these hematologic cancer cells relative to normal leukocytes would potentially represent a differential strategy and perhaps even improve disease outcomes.In this study, we used a combinatorial phage display-based subtractive selection 7-9 to identify ligand motifs that bind to specific cell surface receptors on human leuk...

Therapies selectively targeting ischemic myocardium could be applied by intravenous injection. Here, we report an approach for ischemic tissue-selective targeting based on in vivo screening of random peptide sequences using phage display. We performed in vivo biopanning using a phage library in a rat model of ischemia-reperfusion and identified three peptide motifs, CSTSMLKAC, CKPGTSSYC, and CPDRSVNNC, that exhibited preferential binding to ischemic heart tissue compared to normal heart as well as other control organs. The CSTSMLKAC sequence was capable of mediating selective homing of phage to ischemic heart tissue. The CSTSMLKAC peptide was then made as a fusion protein with Sumo-mCherry and injected intravenously in a mouse model of myocardial ischemia-reperfusion injury; subsequently, bio-distribution of SumomCherry-CSTSMLKAC was measured with quantitative ELISA. The targeting peptide led to a significant increase in homing to ischemic left ventricle compared to tissues from non-ischemic left ventricle, the right ventricle, lung, liver, spleen, skeletal muscle, and brain (all p<0.001). These results indicate that the peptide sequence CSTSMLKAC represents a novel molecular tool that may be useful in targeting ischemic tissue and delivering bioengineered proteins into the injured myocardium by systemic intravenous administration.

Ligand-directed delivery of agents to leukemia and lymphoma cells has the potential to yield new mechanistic disease insights and targeted therapies. Here we set out to target the macropinocytotic pathway with a combinatorial approach. From the screening of acute T-lymphoblastic leukemia Molt-4 cells with a random phage-display peptide library, we isolated a phage displaying the sequence CAYHRLRRC. This peptide contains a lymph node-homing motif (Cys-Ala-Tyr) and a cell-penetrating motif (Arg-Leu-Arg-Arg). Binding of this ligand-directed phage to a large panel of leukemia/lymphoma cells and to patient-derived samples was much higher than to non-leukemia control cells. CAYHRLRRC phage internalization into Molt-4 cells is both energy-and temperature-dependent. Flow cytometry with fluorescein-labeled peptide and endocytosis blocking with specific inhibitors revealed that CAYHRLRRC is indeed taken up through macropinocytosis in Molt-4 and K562 human leukemia cells. Unexpectedly, the cell surface receptor for the CAYHRL-RRC peptide is not a heparan sulfate proteoglycan as it would be predicted for other cell-penetrating peptides. Confirming this interpretation, a CAYHRLRRC-directed peptidomimetic-induced cell death in all the leukemia and lymphoma cells was evaluated, whereas a control transactivator of transcription protein (tat)-directed proapoptotic peptidomimetic was non-selective. In summary, the targeting peptide CAYHRLRRC is selectively internalized through macropinocytosis in leukemia and lymphoma cells and has potential as a drug lead for ligand-directed anti-leukemia therapies.Leukemias and lymphomas are hematological malignant diseases characterized by impaired differentiation, increased clonal cell proliferation, and hematopoiesis suppression; the standard treatment for these tumors today is still predominantly based on nonspecific cytotoxics that disrupt nucleic acid and protein synthesis, often with severe side effects and relatively poor outcomes (1-3). However, selective anti-leukemia drugs have recently been developed (4), thus conceptually validating the scientific hope for a revolutionary targeted pharmacology against this group of diseases.Over the past decade, we have selected phage-display random peptide libraries in vitro and in vivo to isolate and exploit tumor-specific and angiogenesis-related ligand-receptor systems toward targeted drug design and translation (5-7). Because cell trafficking and homing from the blood and/or lymphatic vessels to lymphoid and myeloid tissues to virtually all organs are essential leukocyte functions, we reasoned that targeting membranes would be a suitable approach to discover leukemia-specific ligands. Cell surface-binding peptide motifs have been reported in lymphoma and leukemia lines (8 -11). Unfortunately, so far their corresponding receptors are either unknown (9, 10) or relatively nonspecific adhesion molecules such as certain integrins (8, 11) to which ligand binding does not enable clear enough differentiation between normal leukocytes and tumor cells; ...