The actin cytoskeleton is necessary for cell viability and plays crucial roles in cell motility, endocytosis, growth, and cytokinesis. Hence visualization of dynamic changes in F-actin distribution in vivo is of central importance in cell biology. This has been accomplished by the development of fluorescent protein fusions to actin itself or to various actin-binding proteins, actin cross-linking proteins, and their respective actin-binding domains (ABDs). Although these protein fusions have been shown to bind to F-actin in vivo, we show that the fluorescent protein used for visualization changes the subset of F-actin labeled by an F-actin ABD probe. Further, different amino acid linkers between the fluorescent protein and ABD induced a similar change in localization. Although different linkers and fluorescent proteins can alter the subset of actin bound by a particular ABD, in most cases, the fusion protein did not label all of a cell's F-actin all of the time. Even LimEΔcoil and GFP-actin, which have been used extensively for cytoskeletal visualization, were highly variable in the subsets of actin that they labeled. Lifeact, conversely, clearly labeled cortical F-actin as well as F-actin in the anterior pseudopods of motile cells and in macropinocytotic cups. We conclude that Lifeact most accurately labels F-actin and is the best currently available probe for visualization of dynamic changes in F-actin networks.
This study aimed to investigate the relationship between newly formed lipid droplets and lipid droplet surface proteins, including perilipin, adipose differentiation related protein (ADRP), and p200 kDa protein (p200) in 3T3-L1 preadipocytes, during lipogenesis. Sterol ester was used to induce nascent lipid droplets in 3T3-L1 preadipocytes and the sequence of lipids and lipid droplet surface proteins was studied using a combination of immunohistochemistry and Nile red staining/Oil red O. We demonstrated that, although most growing lipid droplets appeared to have a lipid core surrounded by a fluorescent rim of ADRP, perilipin, and p200, tiny protein aggregates of ADRP, perilipin, or p200 could also be found to occur in the absence of lipid accumulation. In addition, ADRP associated with nascent lipid droplets prior to that of perilipin or p200. We provide evidence that lipid droplet surface proteins, especially ADRP and perilipin, are important in serving as a nucleation center for the assembly of lipid to form nascent lipid droplets.
SummaryBiarsenical-tetracysteine fluorescent protein tagging has been effectively used in a variety of cell types. It has the advantage of requiring a much smaller peptide alteration to existing proteins than fusion to green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP). However, there are no reports of the tetracysteine tagging system being used in Dictyostelium. In order to establish this tagging system in Dictyostelium, the filamin gene (FLN) was modified to express a C-terminal tetracysteine sequence and then transfected into cells. After addition of either FlAsH-EDT 2 or ReAsH-EDT 2 , the fluorescence intensity of cells increased in a time-dependent manner and reached a plateau after 3 h of incubation. ReAsH had a much stronger and more specifically localized fluorescent signal compared with FlAsH. After removal of the ReAsH-EDT 2 reagent, the fluorescence signal remained detectable for at least 24 h. The localization of filamin labelled by ReAsH was similar to that of an FLN-mRFP fusion protein, but the fluorescence signal from the ReAsH-labelled protein was stronger. Our findings suggest that the ReAsHtetracysteine tagging system can be a useful alternative for in vivo protein tagging in Dictyostelium.
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