Cytoplasmic Probing

In the early 20th century, W. H. Lewis, an embryologist at Carnegie Institute of Washington, took time lapse motion pictures of cells grown in culture (Lazarides and Revel, 1979). Lewis treated the fibroblasts (active cells which repair wounds) with protein cleaving enzymes to break up adhesive contacts and networks of extracellular material holding the cells together. He separated the resulting clumps and grew individual cells in a nutrient medium. After a few hours, Lewis' fibroblasts had acquired a polygonal shape. Along the shortest side of the polygon were lamellipodia, delicate sheet-like extensions of the cytoplasm. He exclaimed: "the ruffled lamellipodia slowly bend back and forth like the ruffles of a dress in a slight breeze" (Lazarides and Revel, 1979). Later, Ingram and Abercrombie at University College London studied the membrane coated ruffled lamellipodium, fibroblasts' main locomotion organ. The lamellipodium forms strings and contacts with the substrate on which the cell rests, stretching portions of the rest of the cell among several temporary adhesions. When the cell is spread out to its fullest extent it maintains one or two primary ruffles, each of which tend to lead the cell in a particular direction. The cell then migrates with changes in direction which typically come at intervals of several hours. If a ruffle does not stick and the cell move in another direction, the ruffle folds back on the upper surface of the cell, collapses onto it and disappears into the cell (Figure 5.22, 5.23 and 5.24).

Figure 5.22: Amoeboid movement in a time-lapse series by U. P. Roos at the University of Zurich. 1a) Probing lamellipodia (arrow-heads) in direction „of cell movement. Retraction fiber (arrow) shows trailing end of cell. b) Cytoplasm has flowed to fill out lamellipodia. c) Finger-like filopodia (arrow) probe direction. d) Cytoplasm advances, moving cell. With permission from U. P. Roos (1984).

Figure 5.22: Amoeboid movement in a time-lapse series by U. P. Roos at the University of Zurich. 1a) Probing lamellipodia (arrow-heads) in direction „of cell movement. Retraction fiber (arrow) shows trailing end of cell. b) Cytoplasm has flowed to fill out lamellipodia. c) Finger-like filopodia (arrow) probe direction. d) Cytoplasm advances, moving cell. With permission from U. P. Roos (1984).

Using the electron microscope, Lazarides and Revel (1979) described the lamellipodium as a somewhat irregular sheet of cytoplasm about one tenth of a micrometer (100 nanometers) thick. It is decorated along the edge by small protrusions resembling stubby fingers which attach themselves to the surface of the dish, and can quickly extend several times their initial length into rod-like filopodia. These are highly dynamic structures that can change from a fluid state to a rigid one in less than a second by virtue of the sudden assembly of actin filaments. Growing from the cell surface, they provide spatial guides between which the cell membrane and cytoplasm flows to form the ruffle. The filopodia either attach themselves to the substrate and become rigid, or melt back into the cell. The cell adheres to the surface below it at only a few distinct sites, rather like a hand contacting a table top only with finger tips. Permanent contacts of the ruffle with substrate result in adhesion plaques; as the cell moves the plaques are continuously formed and broken. In addition to their role in cell adhesion and motility, the ruffles and particularly the filopodia serve a sensory function. In the developing embryo when the filopodium of one migrating nerve cell makes contact with the surface of another, the cells stop moving toward each other. Similarly, when the filopodium of a cell spreading in tissue culture touches the surface of a cell that is already flattened, the membrane of the first cell will flow in a direction opposite to the point of contact. This type of sensitive regulation of growth processes is defective in malignant cancer cell.

Figure 5.23: Cytoplasmic organization of a cultured cell moving to the right. Microtubules (MT) radiate from the centrosome (C), located near the nucleus (N), Golgi elements (G), and lysosomes (L). Mitochondria (M) and endoplasmic reticulum (ER) are aligned along MT. Phagocytic and pinocytic vacuoles (P) and secretory vesicles (V) move along MT between the centrosome and periphery. Intermediate filaments (IF) roughly parallel MT. Stress fibers (SF) are the cytoplasmic anchors for extracellular matrix fibers (MF) which contact the substrate. At the leading edge of the moving cell, lamellipodia and filopodia, comprised of actin bundles and meshwork, probe the future. At the trailing end, retraction fibers (RF) contain actin bundles and intermediate filaments. With permission from Marc DeBrabander (1982) and Janssen Pharmaceutics Research Laboratories.

Figure 5.23: Cytoplasmic organization of a cultured cell moving to the right. Microtubules (MT) radiate from the centrosome (C), located near the nucleus (N), Golgi elements (G), and lysosomes (L). Mitochondria (M) and endoplasmic reticulum (ER) are aligned along MT. Phagocytic and pinocytic vacuoles (P) and secretory vesicles (V) move along MT between the centrosome and periphery. Intermediate filaments (IF) roughly parallel MT. Stress fibers (SF) are the cytoplasmic anchors for extracellular matrix fibers (MF) which contact the substrate. At the leading edge of the moving cell, lamellipodia and filopodia, comprised of actin bundles and meshwork, probe the future. At the trailing end, retraction fibers (RF) contain actin bundles and intermediate filaments. With permission from Marc DeBrabander (1982) and Janssen Pharmaceutics Research Laboratories.

Northwestern University biologist Guenter Albrecht-Buehler (1977, 1980) has studied isolated cytoplasm and beholds intelligent behavior. Examples include the movements of single cell organisms alternating between tumbling and smooth motions (Adler, 1969), swimming of motile bacteria in chemotactic environments (Macnab and Koshland, 1972), backward swimming of Paramecium after collision with an object, and the amoeboid directed locomotion of cultured 3T3 mouse cells. Albrecht-Buehler classifies three major types of amoeboid motion: 1) force generation (contraction of an actin-myosin complex, assembly-disassembly of filament polymers, dynein-MT sliding, filament rotation), 2) transmission of motile forces to the framework work of a cell (cytoskeleton in general, anchorage of fibrous elements to the cell surface in particular) and 3) generation of traction (adhesion plaques, extracellular matrix, basement membranes).

Figure 5.24: Cell treated with microtubule assembly inhibiting drug. The cell has lost its polarity. Lamellipodia form around the periphery of the "blob"ular cell. The centrioles (C) are separated from the nucleus. Golgi elements (G), lysosomes (L), mitochondria (M) and endoplasmic reticulum (ER) are disorderly scattered throughout the cytoplasm. With permission from Marc DeBrabander (1982) and Janssen Pharmaceutica Research Laboratories.

Figure 5.24: Cell treated with microtubule assembly inhibiting drug. The cell has lost its polarity. Lamellipodia form around the periphery of the "blob"ular cell. The centrioles (C) are separated from the nucleus. Golgi elements (G), lysosomes (L), mitochondria (M) and endoplasmic reticulum (ER) are disorderly scattered throughout the cytoplasm. With permission from Marc DeBrabander (1982) and Janssen Pharmaceutica Research Laboratories.

Albrecht-Buehler further divides the control of cell migration into three subtopics: 1) generation of a front-rear axis (polarity involves MTOC and MT), 2) cell body coordination-how the forty billion protein molecules comprising each cell act cooperatively to move as a unit, and 3) logic of migration-the rules of changing direction. Migrating cells make decisions and globally assess their environment. Albrecht-Buehler asserts that the control of amoeboid cell migration provides evidence of cytoplasmic intelligence because it requires coordination of many cell domains and navigation which involves assessment of the environment.

But the cell is not the indivisible unit of intelligent behavior. Albrecht-Buehler contends there are hierarchical strata of information processing within cells. He has shown that small fragments of cytoplasm comprising about two percent of cell volume can be torn out of living fibroblasts (Albrecht-Buehler, 1980). These "microplasts" are devoid of any genetic input, yet can move filopodia, ruffle lamellipodia and produce blebs. Some fragments are so small they appear as isolated active filopodia, ruffles, or blebs and suggest that cells contain cytoplasmic domains capable of autonomous amoeboid movement. Albrecht-Buehler asserts that these cytoplasmic activities within cells are controlled by a superior stratum of organization to permit coherent locomotion. This level of control is not located in the genes since cells with their nuclei removed still exhibit migration behavior. There is intelligence in the cytoplasm. The cell has a driver's license.

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