Expression of Genetically Encoded Fluorescent Probes to Monitor Phospholipid Dynamics in Live Neutrophils

Microinjection of fluorescent probes into living plant cells reveals new aspects of cell structure and function. Microtubules and actin filaments are dynamic components of the cytoskeleton and are involved in cell growth, division and intracellular transport. To date, cytoskeletal probes used in microinjection studies have included rhodamine-phalloidin for labelling actin filaments and fluorescently labelled animal tubulin for incorporation into microtubules. From a recent study of Tradescantia stamen hair cells it appears that actin may have a role in defining the plane of cell division. Unlike microtubules, actin is present in the cell cortex and delimits the division site throughout mitosis. Herein, I shall describe actin, its arrangement and putative role in cell plate placement, in another material, living cells of Tradescantia leaf epidermis.The epidermis is peeled from the abaxial surface of young leaves usually without disruption to cytoplasmic streaming or cell division. The peel is stuck to the base of a well slide using 0.1% polyethylenimine and bathed in a solution of 1% mannitol +/− 1 mM probenecid.

Download Full-text

Live Confocal Analysis of Fertilization and Early Development

Microscopy and Microanalysis ◽

10.1017/s1431927600031135 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 1012-1013

Author(s):

Uyen Tram ◽

William Sullivan

Keyword(s):

Drosophila Melanogaster ◽

Embryonic Development ◽

Real Time ◽

Early Development ◽

Fluorescent Probes ◽

Primary Defect ◽

Fluorescent Analysis ◽

Dynamic Event ◽

Enormous Amount ◽

Fluorescent Studies

Embryonic development is a dynamic event and is best studied in live animals in real time. Much of our knowledge of the early events of embryogenesis, however, comes from immunofluourescent analysis of fixed embryos. While these studies provide an enormous amount of information about the organization of different structures during development, they can give only a static glimpse of a very dynamic event. More recently real-time fluorescent studies of living embryos have become much more routine and have given new insights to how different structures and organelles (chromosomes, centrosomes, cytoskeleton, etc.) are coordinately regulated. This is in large part due to the development of commercially available fluorescent probes, GFP technology, and newly developed sensitive fluorescent microscopes. For example, live confocal fluorescent analysis proved essential in determining the primary defect in mutations that disrupt early nuclear divisions in Drosophila melanogaster. For organisms in which GPF transgenics is not available, fluorescent probes that label DNA, microtubules, and actin are available for microinjection.

Download Full-text