Lipoprotein-mediated delivery of BODIPY-labeled sterol and sphingolipid analogs reveals lipid transport mechanisms in mammalian cells

Lipids are often introduced into cell membranes directly from solvent or from lipophilic artificial carriers, such as cyclodextrins. A physiological lipid entry route into mammalian cells is via lipoprotein mediated uptake. In this review, we discuss the introduction of BODIPY-labeled sterol and sphingolipid analogs into mammalian cells via highor low-density lipoproteins, and the novel findings made by using this strategy. Lipoprotein mediated delivery favors endocytic uptake and initial incorporation of the lipid into membranes of the endosomal compartments. This routing can therefore highlight physiological mechanisms of lipid entry into and exit from the endo-lysosomal membrane system. The underlying principles are of key importance for instance in controlling plasma cholesterol levels and in the development and regression of lysosomal lipid storage diseases. A common denominator for the BODIPY-labeled lipid analogs discussed in this review is that they were synthesized by late Robert Bittman, whose scientific impact radiates far beyond his lifework in organic chemistry. Foreword With the advent of green fluorescent protein, molecular cell biology entered a new era of live cell imaging. Yet, in the cellular context at least half of the proteins are either stably or dynamically associated with membranes, and visualizing lipids is equally relevant. Tagging of lipids with fluorophores is more complicated than that of proteins. This is not only because fluorescent lipids cannot be prepared simply by cloning but also because of the typically large size and hydrophilic nature of fluorophores relative to lipids and hence, their potentially substantial disturbing effects. The choice, positioning and orientation of the fluorescent moiety are relevant for the biophysical nature and metabolic processing of the lipid. On the other hand, the fluorophore choice depends on its optical properties, such that it can be sensitively detected with available microscopy systems and without excessive phototoxicity for living cells. One of the widely used, relatively hydrophobic fluorophores with high quantum yield and photostability is the dipyrrometheneboron difluoride chelate (4,4,-difluoro4-bora-3a,4a-diaza-s-indacene), BODIPY. The development of suitable lipid probes for imaging necessitates a smooth co-operation between the synthesis and application ends, so that several rounds of trials (and errors) can be swiftly executed. Such interdisciplinary collaborations were inherent for Robert Bittman, to whom this Special Issue of Chemistry and Physics of Lipids is dedicated. In this review, we will highlight some of the findings made together with Robert, by taking advantage of the BODIPY-labeled sterol and sphingolipid analogs developed by him. Our collaboration with Robert Bittman was initiated in 2006, yielding the first report on the use and characteristics of BODIPY-cholesterol (Fig. 1) in living cells (Hölttä-Vuori et al., 2008). This compound was initially synthesized and characterized in model membranes by Bittman and coworkers (Li and Bittman, 2007; Shaw et al., 2006). We will briefly review our early observations on the properties of BODIPY-cholesterol and its features in cells and tissues. Please note that several aspects related to BODIPY-cholesterol, such as its lateral diffusion in membranes, use for diaminobenzidine (DAB) photooxidation enabling visualization by electron microscopy, and for tracking sterol routing from endocytic recycling compartment to the plasma membrane, are discussed by Daniel Wustner and Herbert Stangl in this Issue. For additional information on BODIPY-cholesterol, we refer the reader to this chapter. In the present article, the main focus is on strategies where BODIPY-cholesterol or BODIPY-sphingolipid analogs have been introduced into cells via lipoprotein particles; the rationale being that this represents a major physiological lipid entry mechanism in mammalian cells (Ikonen, 2008). Most of the work discussed deals with experiments using low-density lipoprotein (LDL) particles as vehicles. We will also briefly discuss BODIPY-lipid uptake via high-density lipoproteins (HDL), representing a physiological lipid uptake route in hepatocytes and steroidogenic cells (Ikonen, 2008). In addition to BODIPY-cholesterol, the sterol analogs discussed here include fatty acid (oleate or linoleate) esters of BODIPYcholesterol and the ketone derivative BODIPY-cholestenone. In all of these, the BODIPY moiety is linked to the carbon-24 of the cholesterol side chain, similarly as in BODIPY-cholesterol (Fig. 1). For BODIPY-sphingolipids, we will review compounds characterized by us in collaboration with Bittman, namely BODIPYFTY720, BODIPY-sphingosine, BODIPY-ceramide as well as the cross-linkable benzophenone-BODIPY-ceramide (Fig. 2).