Actin filaments (F-actin) are protein polymers that undergo rapid assembly and disassembly and control an enormous variety of cellular processes ranging from force production to regulation of signal transduction. Consequently, imaging of F-actin has become an increasingly important goal for biologists seeking to understand how cells and tissues function. However, most of the available means for imaging F-actin in living cells suffer from one or more biological or experimental shortcomings. Here we describe fluorescent F-actin probes based on the calponin homology domain of utrophin (Utr-CH), which binds F-actin without stabilizing it in vitro. We show that these probes faithfully report the distribution of F-actin in living and fixed cells, distinguish between stable and dynamic F-actin, and have no obvious effects on processes that depend critically on the balance of actin assembly and disassembly.

Anthocyanins are flavonoid pigments synthesized in the cytoplasm and stored inside vacuoles. Many plant species accumulate densely packed, 3-to 10-mm diameter anthocyanin deposits called anthocyanin vacuolar inclusions (AVIs). Despite their conspicuousness and importance in organ coloration, the origin and nature of AVIs have remained controversial for decades. We analyzed AVI formation in cotyledons of different Arabidopsis thaliana genotypes grown under anthocyanin inductive conditions and in purple petals of lisianthus (Eustoma grandiorum). We found that cytoplasmic anthocyanin aggregates in close contact with the vacuolar surface are directly engulfed by the vacuolar membrane in a process reminiscent of microautophagy. The engulfed anthocyanin aggregates are surrounded by a single membrane derived from the tonoplast and eventually become free in the vacuolar lumen like an autophagic body. Neither endosomal/prevacuolar trafficking nor the autophagy ATG5 protein is involved in the formation of AVIs. In Arabidopsis, formation of AVIs is promoted by both an increase in cyanidin 3-O-glucoside derivatives and by depletion of the glutathione S-transferase TT19. We hypothesize that this novel microautophagy mechanism also mediates the transport of other flavonoid aggregates into the vacuole.

Contractile arrays of actin filaments (F-actin) and myosin-2 power diverse biological processes. Contractile array formation is stimulated by the Rho GTPases Rho and Cdc42; after assembly, array movement is thought to result from contraction itself. Contractile array movement and GTPase activity were analyzed during cellular wound repair, in which arrays close in association with zones of Rho and Cdc42 activity. Remarkably, contraction suppression prevents translocation of F-actin and myosin-2 without preventing array or zone closure. Closure is driven by an underlying “signal treadmill” in which the GTPases are preferentially activated at the leading edges and preferentially lost from the trailing edges of their zones. Treadmill organization requires myosin-2 powered contraction and F-actin turnover. Thus, directional gradients in Rho GTPase turnover impart directional information to contractile arrays and proper functioning of these gradients is dependent on both contraction and F-actin turnover.

Increased breast density attributed to collagen I deposition is associated with a 4–6 fold increased risk of developing breast cancer. Here, we assessed cellular metabolic reprogramming of mammary carcinoma cells in response to increased collagen matrix density using an in vitro 3D model. Our initial observations demonstrated changes in functional metabolism in both normal mammary epithelial cells and mammary carcinoma cells in response to changes in matrix density. Further, mammary carcinoma cells grown in high density collagen matrices displayed decreased oxygen consumption and glucose metabolism via the tricarboxylic acid (TCA) cycle compared to cells cultured in low density matrices. Despite decreased glucose entry into the TCA cycle, levels of glucose uptake, cell viability, and ROS were not different between high and low density matrices. Interestingly, under high density conditions the contribution of glutamine as a fuel source to drive the TCA cycle was significantly enhanced. These alterations in functional metabolism mirrored significant changes in the expression of metabolic genes involved in glycolysis, oxidative phosphorylation, and the serine synthesis pathway. This study highlights the broad importance of the collagen microenvironment to cellular expression profiles, and shows that changes in density of the collagen microenvironment can modulate metabolic shifts of cancer cells.