Research article
Cytoskeletal scaffolds regulate riboflavin endocytosis and recycling in placental trophoblasts

https://doi.org/10.1016/j.jnutbio.2006.01.008Get rights and content

Abstract

Microfilaments and microtubules (MT) play a vital role in cellular endocytic processes. The present study evaluates the role of these cytoskeletal elements in the apical internalization and postendocytic fate of riboflavin (RF) in placental trophoblasts (BeWo cells). Biochemical modification of the actin and microtubule network by (1) okadaic acid (OA), which disrupts MT-based vesicular trafficking; (2) cytochalasin D and latrunculin B, which promote actin depolymerization; and (3) 2,3-butanedione monoxime (BDM), which inhibits myosin–actin interaction, was confirmed by immunofluorescence microscopy using actin- and tubulin-specific antibodies. Furthermore, involvement of the molecular motors dynein and kinesin was assessed in the presence of (1) sodium orthovanadate, which inhibits dynein-ATPase activity and (2) adenosine 5′-(β,γ-imido)triphosphate tetralithium salt hydrate, a non-hydrolyzable ATP analog, which results in defective kinesin-driven processes. RF internalization consequent to cytoskeletal alterations was compared with that of a clathrin-dependent endocytic marker ([125I]-transferrin [TF]), a caveolae-mediated endocytic substrate ([3H]-folic acid [FA]), and a fluid-phase endocytic marker ([125I]-horse radish peroxidase [HRP]). Apical recycling and bidirectional transport of RF and TF was measured following cytoskeletal alterations. Results indicate that uptake of RF, TF, FA and HRP are markedly reduced (~30–65%) in the presence OA and BDM, suggesting differential sensitivities to modification of kinesin-driven microtubules. However, actin depolymerization negatively affected HRP endocytosis alone, while RF, FA and TF internalization remained unchanged. Disturbances in protein phosphorylation cascades also influenced apical recycling while net ligand transport across monolayers remained unaffected. In conclusion, apical RF trafficking in placental cells is tightly regulated by microtubules and supported by accessory actin involvement.

Introduction

Vitamin B2, commonly referred to as riboflavin (RF), is pivotal to cellular metabolic function and is obtained solely from dietary sources. Flavoproteins utilize flavin mononucleotide and flavin adenine dinucleotide cofactors derived from the vitamin precursor to catalyze redox exchanges in cellular metabolic processes. Deficiency of this vitamin has been evidenced as either a causal or risk factor for anemia, cardiovascular disease and neuro-degenerative disorders [1]. Consequently, delineating the physiological process that maintains a balanced nutritional load within the cell is of paramount significance.

Vectorial transport systems such as carriers, channels, receptors and endocytic vesicles mediate restrictive entry of extracellular nutrients and macromolecules across the plasma membrane in response to physiological stimuli. Unlike carriers and channels that facilitate the uptake of smaller solutes, endocytosis is a eukaryotic process that moves larger cargo such as nutrients, lipids, receptors and bacterial and viral pathogens [2], thereby offering a viable target for delivery of protein-, peptide- and DNA-based entities. Biochemical and morphological studies from our laboratory have demonstrated the existence of a high affinity, temperature-dependent saturable process that follows a classical receptor-mediated endocytic (RME) pathway in the absorption of this water-soluble micronutrient [3], [4], [5]. The endocytic processing of ligands progresses through chronological events of receptor recognition, sequestration into pits, vesicle assembly and scission and, finally, intracellular sorting [6]. Recent studies have shown that this highly ordered vesicular process integrates multiple structural and signaling networks to mediate transfer of selective ligands into cellular domains [6], [7], [8]. Furthermore, processes such as transcytosis or apical recycling that determine the postendocytic fate of the internalized ligands are also extensively regulated by the distributed networks of microtubules and microfilaments [9], [10], [11], [12]. Consequently, trafficking of the vesicle-sequestered RF from early endosomes to late endosomes, recycling endosomes and lysosomes, as well as transcytotic transfer across the cell, requires matrices that would facilitate their spatial sorting.

Structurally, microtubules and microfilaments define the cellular architecture and provide a “scaffold” for shuttling of endocytic cargo [13], [14]. Directional movement along the scaffold is fueled by localized motor proteins, viz., microtubule-based kinesins and dyneins and actin-based myosins [15], [16], [17]. Dyneins and kinesins associate with filamentous microtubules and trigger rapid and bidirectional motility of endocytic vesicles, while myosin motors drive endocytic or exocytic movement along actin filaments underlying the plasma membrane [18], [19].

The objective of the ensuing study was to examine the role of the microtubule and actin networks in apical endocytosis of RF in placental trophoblasts (BeWo) via use of selective modifiers of these cytoskeletal elements. Subsequent morphological changes were evaluated by immunofluorescent staining using actin- and tubulin-specific antibodies, while changes in the motor proteins, dynein and kinesin were measured by Western blot analyses. Structural changes of the cytoskeleton were then correlated to the changes in the endocytic internalization of [3H]-RF and compared with clathrin-dependent [125I]-transferrin (TF), caveolae-mediated [3H]-folic acid (FA) and fluid-phase [125I]-horse radish peroxidase (HRP) endocytosis. Finally, apical recycling and transcytosis of the receptor-mediated ligands, RF and TF, were assessed as a function of alterations to these cytoskeletal elements.

Section snippets

Materials

Riboflavin-[3H(G)] (25 Ci/mmol), holo-TF, 2,3-butanedione monoxime, cytochalasin D, sodium orthovanadate, adenosine 5′-(β,γ-imido)triphosphate tetralithium salt hydrate (AMP-PNP) and monoclonal anti-β-actin were obtained from Sigma (St. Louis, MO, USA). [3H]-FA (25 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). Rabbit polyclonal anti-β-tubulin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Okadaic acid, latrunculin B and HRP were purchased

Okadaic acid-induced effects on tubulin alter receptor- and caveolae-mediated endocytosis

Okadaic acid (OA), a serine-threonine protein phosphatase (PP) inhibitor is specific to PP1 and PP2A. Decreased phosphatase activity has been indicated in microtubule (MT) depolymerization-induced reduction in vesicle based transport [20]. BeWo cells were treated with 0.1–1 μM OA for 30 and 60 min, respectively, and its effect specific to endocytic uptake of RF, FA, TF and HRP was evaluated. Fig. 1A–C revealed a dose-dependent decrease in the apical internalization of RF, FA and TF,

Discussion

Endocytic processes are described by complex events requiring extensive structural links within the cell. Our laboratory has proposed such a receptor-mediated internalization mechanism in the placental and intestinal uptake of RF [3], [4], although the identity of the process mediators remains largely unknown. In this study, we examine the dependence of RF trafficking on cytoskeletal networks of microtubules and cortical actin in placental trophoblasts.

Microtubule disruption induced by okadaic

References (29)

  • M.A. Phelps et al.

    Cytoskeletal motors and cargo in membrane trafficking: opportunities for high specificity in drug intervention

    Drug Discov Today

    (2003)
  • S.N. Huang et al.

    Involvement of a receptor-mediated component in cellular translocation of riboflavin

    J Pharmacol Exp Ther

    (2000)
  • S.N. Huang et al.

    Riboflavin uptake in human trophoblast-derived BeWo cell monolayers: cellular translocation and regulatory mechanisms

    J Pharmacol Exp Ther

    (2001)
  • S.N. Huang et al.

    Involvement of endocytic organelles in the subcellular trafficking and localization of riboflavin

    J Pharmacol Exp Ther

    (2003)
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    Funding Source: This study was supported by funds from the National Institutes of Health (Grant DK56631).

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