Dynamic membrane remodeling at invadopodia differentiates invadopodia from podosomes

Abstract

Invadopodia are specialized actin-rich protrusions of metastatic tumor and transformed cells with crucial functions in ECM degradation and invasion. Although early electron microscopy studies described invadopodia as long filament-like protrusions of the cell membrane adherent to the matrix, fluorescence microscopy studies have focused on invadopodia as actin-cortactin aggregates localized to areas of ECM degradation. The absence of a clear conceptual integration of these two descriptions of invadopodial structure has impeded understanding of the regulatory mechanisms that govern invadopodia. To determine the relationship between the membrane filaments identified by electron microscopy and the actin-cortactin aggregates of invadopodia, we applied rapid live-cell high-resolution TIRF microscopy to examine cell membrane dynamics at the cortactin core of the invadopodia of human carcinoma cells. We found that cortactin docking to the cell membrane adherent to 2D fibronectin matrix initiates invadopodium assembly associated with the formation of an invadopodial membrane process that extends from a ventral cell membrane lacuna toward the ECM. The tip of the invadopodial process flattens as it interacts with the 2D matrix, and it undergoes constant rapid ruffling and dynamic formation of filament-like protrusions as the invadopodium matures. To describe this newly discovered dynamic relationship between the actin-cortactin core and invadopodial membranes, we propose a model of the invadopodial complex. Using TIRF microscopy, we also established that - in striking contrast to the invadopodium - membrane at the podosome of a macrophage fails to form any process- or filament-like membrane protrusions. Thus, the undulation and ruffling of the invadopodial membrane together with the formation of dynamic filament-like extensions from the invadopodial cortactin core defines invadopodia as invasive superstructures that are distinct from the podosomes.

Figure 1

Cell membrane dynamics at invadopodia. Human breast carcinoma wt c-Src MDA-MB-231 cells expressing GFP-Cortactin and membrane marker IL2R-mCherry were cultured on a layer of FN. A. Initiation of invadopodium formation. An invadopodium in early stages of formation is shown selected with a region-of-interest (ROI) frame. The ROI montage shows every 6^th frame, and the total duration of the time-lapse image acquisition used for the montage is 6.9 min. Scale bar indicates 3 μm. B. Mature invadopodium. This stage is characterized by a high degree of cell membrane morphological changes as exemplified by the invadopodial complex analyzed in the ROI. The montage of ROIs shows every 5^th frame, and the total duration of the time-lapse image acquisition used for the montage is 5.3 min. Scale bar indicates 3 μm. C. Enlarged views of three frames from the ROI montage shown in B. White arrows point to filament-like membrane extensions that originate from the cortactin core; they also indicate the furthest extent of cortactin along the membrane filament-like protrusion. Yellow arrowheads point to membrane ruffles.

Figure 2

A. Dynamics of the cell membrane at the focal adhesion of HFF transiently expressing GFP-Vinculin and membrane marker IL2R-mCherry. HFF were cultured on a 2D FN matrix. A representative focal adhesion is highlighted by the ROI. The ROI montage shows every 10^th frame of the time lapse of focal adhesion extension; the total duration of time-lapse image acquisition used for the montage is 12.7 min. Scale bar indicates 3 μm. B. Dynamics of the cell membrane at a podosome of IC-21 macrophage transiently expressing GFP-Cortactin and membrane marker IL2R-mCherry. Transfected macrophages were cultured on a 2D FN matrix. The ROI montage shows every 10^th frame, and the total duration of the time-lapse image acquisition used for the montage is 5.2 min. Scale bar indicates 1 μm. C. TIRF microscopy of IC-21 macrophage expressing IL2R-mCherry and immuno-labeled with phalloidin-Alexa488 and anti-vinculin Cy-5-conjugated antibody. Enlargements of the ROI are shown as insets.

Figure 3

A, Model of the invadopodial complex. Dashed line indicates the upper boundary of the membrane-cytoplasmic zone visualized by TIRF microscopy. B, Invadopodial complex formation in wt c-Src MDA-MB-231 cells invading 3D matrix of 3 mg/ml rat tail collagen type I fluorescently labeled with Alexa647. MDA-MB-231 cells transiently expressing GFP-Cortactin and IL2R-mCherry were polymerized between two layers of rat tail collagen and allowed to invade 3D collagen matrix overnight. White arrows point to cortactin-rich invadopodial cores and yellow arrow points to filament-like invadopodia. Scale bar indicates 10 μm.