Delivery of Exogenous Imaging Probes for Cellular Microscopy
thesisposted on 25.10.2015, 00:00 authored by Shabnam Mohandessi
Optical microscopy has emerged as one of the most important methods for studying living cells and their molecular components. Such studies require fluorescent probes that either selectively label functional biomolecules, thereby allowing dynamic tracking of their sub-cellular location, or else partition to a sub-cellular domain and report particular chemical or physical properties such as pH, viscosity or calcium ion concentration. While genetically encoded fluorescent proteins are the most common probe type, there has been considerable interest in developing organic fluorophores, metal complexes and inorganic nanoparticles for use in live cell imaging studies. These exogenous probes can be synthetically modified to be brighter, more resistant to photobleaching or more analytically responsive than their fluorescent protein analogs. However, a key limitation of many fluorescent species is that they are often impermeant to cell membranes and are therefore difficult or impossible to use as probes for live cell imaging studies. This dissertation describes methods that can be used to deliver membrane-impermeant, fluorescent probes into the cytoplasm of cultured mammalian cells for microscopic imaging of protein function. Specifically, methods are described to deliver luminescent terbium complexes with long emission lifetimes that selectively label recombinant fusion proteins in cellulo. Heterodimers of a 2-hydroxyisophthalamide terbium macrocycle (Lumi4-Tb) linked to the antibiotic trimethoprim (TMP) were observed to selectively label Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins inside cells following cytoplasmic delivery. Cellular uptake and specific labeling of eDHFR were visualized using time-gated microscopy of long-lived (~ms) terbium emission or Förster resonance energy transfer (FRET) between terbium and fluorescent proteins. Chapter 1 provides an overview of luminescent probe types and intracellular delivery methods that have been described in the literature. Chapter 2 describes in detail physical and biochemical methods that breach the lipid bilayer in order to deliver terbium labels into the cytoplasm including microinjection, reversible membrane permeabilization with the bacterial toxin streptolysin-O and osmotic lysis of macropinocytic vesicles. In chapter 3, the successful cytoplasmic delivery of Lumi4 and TMP-Lumi4 conjugated to arginine-rich, cell penetrating peptides (CPPs) is described. Conjugates of nonaarginine and HIV-tat-derived sequences were observed to directly translocate into the cytoplasm of various cell types. CPP-mediated delivery should be adaptable for delivery of a wide variety of small molecule probes, and Chapter 4 outlines recent data and ongoing research in the Miller laboratory that aims to quantitatively understand the factors that control translocation of CPP-probe conjugates from extracellular medium into the cytoplasm of live cells.