Increasing evidence supports a critical role for the septin cytoskeleton at the plasma membrane during physiological processes including motility, formation of dendritic spines or cilia, and phagocytosis. We sought to determine how septins regulate the plasma membrane, focusing on this cytoskeletal element's role during effective amoeboid motility. Surprisingly, septins play a reactive rather than proactive role, as demonstrated during the response to increasing hydrostatic pressure and subsequent regulatory volume decrease. In these settings, septins were required for rapid cortical contraction, and SEPT6-GFP was recruited into filaments and circular patches during global cortical contraction and also specifically during actin filament depletion. Recruitment of septins was also evident during excessive blebbing initiated by blocking membrane trafficking with a dynamin inhibitor, providing further evidence that septins are recruited to facilitate retraction of membranes during dynamic shape change. This function of septins in assembling on an unstable cortex and retracting aberrantly protruding membranes explains the excessive blebbing and protrusion observed in septin-deficient T cells.

Multiphoton microscopy has become staple tool for tracking cells within tissues and organs due to superior depth of penetration, low excitation volumes, and reduced phototoxicity. Many factors, ranging from laser pulse width to relay optics to detectors and electronics, contribute to the overall ability of these microscopes to excite and detect fluorescence deep within tissues. However, we have found that there are few standard ways already described in the literature to distinguish between microscopes or to benchmark existing microscopes to measure the overall quality and efficiency of these instruments. Here, we discuss some simple parameters and methods that can either be used within a multiphoton facility or by a prospective purchaser to benchmark performance. This can both assist in identifying decay in microscope performance and in choosing features of a scope that are suited to experimental needs.