Cancer initiation and progression follow complex molecular and structural changes in the extracellular matrix and cellular architecture of living tissue. However, it remains poorly understood how the transformation from health to malignancy alters the mechanical properties of cells within the tumour microenvironment. Here, we show using an indentation-type atomic force microscope (IT-AFM) that unadulterated human breast biopsies display distinct stiffness profiles. Correlative stiffness maps obtained on normal and benign tissues show uniform stiffness profiles that are characterized by a single distinct peak. In contrast, malignant tissues have a broad distribution resulting from tissue heterogeneity, with a prominent low-stiffness peak representative of cancer cells. Similar findings are seen in specific stages of breast cancer in MMTV-PyMT transgenic mice. Further evidence obtained from the lungs of mice with late-stage tumours shows that migration and metastatic spreading is correlated to the low stiffness of hypoxia-associated cancer cells. Overall, nanomechanical profiling by IT-AFM provides quantitative indicators in the clinical diagnostics of breast cancer with translational significance.

Macrophages are an important component of the innate immune response and their ability to migrate toward pathogens is critical to their success as first responders. Several proteins are known to contribute to the migration of macrophages and their roles have been studied using Boyden chambers, Dunn chambers, and micropipette pointsources. However, visualizing macrophage migration has been difficult due to their inherently strong adhesion. We have been able to study macrophage migration in two dimensions using a novel surface preparation in which PDMS coated coverslips are stamped with the extracellular matrix protein fibronectin. LR5 macrophages were individually tracked on these surfaces in the presence of CSF-1, a physiologically relevant chemokine, using time-lapse microscopy. Analysis of the trajectories of motion has allowed us to describe motility in terms of speed, persistence time, and the random motility coefficient. LR5 cells in which endogenous Cdc42 or WASp was reduced using small interfering RNA were also examined to determine the role of these proteins in migration. Our results indicate that reduction of WASp levels leads to a significant reduction in motility. The knockdown of Cdc42 leads to a reduction in the random motility coefficient and sensitivity to ligand chemistry. Our results are in contrast to other studies which have linked Cdc42 and WASp to directional sensing but have found no defect in random motility. The roles of PI3K in macrophage migration are currently being investigated. We are also beginning to study the forces macrophages produce while migrating using microfabricated post array detectors. The cell lines with reduced protein levels will also be studied on posts to determine the role of each protein in force production.

Biointerfaces capable of biological recognition and specificity are sought after for conferring bioinspired functionality onto synthetic biomaterials systems. This is important for biosensing, bioseparations, and biomedical materials. Here, we demonstrate how intrinsic polymer-protein interactions between highly localized polyethylene glycol (PEG) brushes and PEG-binding antibodies can be used for sorting specific biomolecules from complex bulk biological fluids to synthetic nanoscale targets. A principal feature lies with the antifouling property of PEG that prevents unspecific binding. Exclusive access is provided by anti-PEG, which acts as a biohybrid molecular adaptor that sifts out and targets specific IgG "cargo" from solution to the PEG. The PEG can be reversibly washed and targeted in blood serum, which suggests potential benefits in technological applications. Moreover, anti-PEG binding triggers a stimuli-responsive conformational collapse in the PEG brush, thereby imparting an intrinsic "smart" biorecognition functionality to the PEG that can considerably impact its use as an antifouling biomaterial.

Two distinct dimers are formed during the initial steps of actin polymerization. The first one, referred to as the 'lower dimer' (LD) was discovered many years ago by means of chemical crosslinking. Owing to its transient nature, a biological relevance had long been precluded when, using LD-specific antibodies, we detected LD-like contacts in actin assemblies that are associated with the endolysosomal compartment in a number of different cell lines. Moreover, immunofluorescence showed the presence of LD-related structures at the cell periphery of migrating fibroblasts, in the nucleus, and in association with the centrosome of interphase cells. Here, we explore contributions of the LD to the assembly of supramolecular actin structures in real time by total internal reflection fluorescence (TIRF) microscopy. Our data shows that while LD on its own cannot polymerize under filament forming conditions, it is able to incorporate into growing F-actin filaments. This incorporation of LD triggers the formation of X-shaped filament assemblies with barbed ends that are pointing in the same direction in the majority of cases. Similarly, an increased frequency of junction sites was observed when filaments were assembled in the presence of oxidized actin. This data suggests that a disulfide bridge between Cys374 residues might stabilize LD-contacts. Based on our findings, we propose two possible models for the molecular mechanism underlying the supramolecular actin patterning in LD-related structures.

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