Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions <em>In Vivo</em>

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

University of Wisconsin-Madison

Cellular damage triggers rapid resealing of the plasma membrane and repair of the cortical cytoskeleton. Plasma membrane resealing results from calcium-dependent fusion of membranous organelles and the plasma membrane at the site of the damage. Cortical cytoskeletal repair results from local assembly of actin filaments (F-actin), myosin-2 and microtubules into an array that closes around the original wound site. Control of the cytoskeletal response is exerted by local activation of the small GTPases, Rho and Cdc42. Recent work has given insight into both the membrane fusion and cytoskeletal responses to plasma membrane damage and we propose that Rho GTPase activation results at least in part from the events that drive membrane repair.

Rehabilitation and the single cell.

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.

Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin.

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.

A Rho GTPase signal treadmill backs a contractile array.

Anthocyanins are flavonoid pigments synthesized in the cytoplasm and stored inside vacuoles. Many plant species accumulate densely packed, 3- to 10-μm 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.

Anthocyanin Vacuolar Inclusions Form by a Microautophagy Mechanism.

Quantitative data is presented that shows significant changes in cellular metabolism in a head and neck cancer cell line 30 min after irradiation. A head and neck cancer cell line (UM-SCC-22B) and a comparable normal cell line, normal oral keratinocyte (NOK) were each separately exposed to 10 Gy and treated with a control drug for disrupting metabolism (potassium cyanide; KCN). The metabolic changes were measured live by fluorescence lifetime imaging of the intrinsically fluorescent intermediate metabolite nicotinamide adenosine dinucleotide (NADH) fluorescence; this method is sensitive to the ratio of bound to free NADH. The results indicated a prompt shift in metabolic signature in the cancer cell line, but not in the normal cell line. Control KCN treatment demonstrated expected metabolic fluxes due to mitochondrial disruption. The detected radiation shift in the cancer cells was blunted in the presence of both a radical scavenger and a HIF-1α inhibitor. The HIF-1α abundance as detected by immunohistochemical staining also increased substantially for these cancer cells, but not for the normal cells. This type of live-cell metabolic monitoring could be helpful for future real-time studies and in designing adaptive radiotherapy approaches.

Brian Burkel

Department of Cell and Regenerative Biology,
Laboratory for Optical and Computational Instrumentation

University of Wisconsin-Madison

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Brian Burkel

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell and Regenerative Biology,Laboratory for Optical and Computational Instrumentation

University of Wisconsin-Madison

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Department of Cell and Regenerative Biology,
Laboratory for Optical and Computational Instrumentation