2.3.1 General Aspects and Possible Mechanisms of Cellular Spreading
In this section, possible mechanisms for cellular spreading, which may be applica-ble to our findings, are detailed. However, there are many more models existing in the literature which are not discussed here for the sake of clarity.
The findings described below have been obtained in studies of spreading of im-mortalized embryonic mouse fibroblasts and show that actin polymerization at the outer edge of the cell and rearward actin movement govern spreading [31, 42, 43].
It has been shown for several cell types that actin polymerization occurs predomi-nantly near the cell membrane while the actin network inside lamellipodia moves rearward [75, 101, 102]. Furthermore, the speed of protrusion of the cell’s outer edge shows a negative correlation with the rate of rearward actin movement [14].
Giannone et al. [42] and Dubin-Thaler et al. [31] found two different spreading behaviors in immortalized embryonic mouse fibroblasts which, however, have no influence on the final cell area or final morphology [31]. Anisotropic spreading in fibroblasts involves filopodia, while isotropically spreading fibroblasts show no filopodia [42]. Furthermore, in isotropically spreading fibroblasts little or no membrane ruffling is observed, while anisotropically spreading fibroblasts do show membrane ruffles [31]. The speed of actin rearward movement increases for isotropic spreading when the cell has nearly completely spread [31]. For anisotropic spreading the speed of actin rearward movement is at a constant high level (similar to that of isotropic spreading at the endphase of spreading) from the onset of spreading [31]. Furthermore, there are indications for a non-constant actin polymerization [31]. Thus, protrusion of the cell edge is thought to be dic-tated by the speed of actin polymerization and the speed of rearward movement of the actin network [31].
Isotropic spreading for the same cell type shows a three-phased spreading behav-ior [42]. In the beginning, fibroblasts spread fast at a constant rate that decreases in the second spreading phase [42]. During the second phase, the fibroblasts show periodic interruptions/retractions and the next and last phase is characterized by either a transition to anisotropic spreading behavior or maintenance of isotropic spreading [42]. The periodic interruptions/retractions are explained by an in-crease in rearward actin movement [42]. Thus, Dubin-Thaleret al. [31] and Gian-noneet al. [42] showed that differences in actin rearward movement can account
Chapter 2 STATE OF THE ART
for different spreading behaviors in one cell type.
A mechanism to explain periodic retractions as well as membrane ruffling in spreading was proposed by Giannone et al. [43]. In this proposed mechanism, myosin activity leads to a rearward force (towards the cell center) being applied on the lamellipodium [43]. These forces lead to upwards bending of the lamel-lipodium as well as formation of adhesion sites at the lamellipodial tip [43]. Sub-sequently, the lamellipodium detaches from the plasma membrane leaving the machinery for actin polymerization in place [43]. Thus, newly polymerizing actin can reconstitute the lamellipodium network [43]. A new cycle of edge retraction can be initiated by the rearward moving actin in the lamellipodium that reaches the myosin in the back of the lamellipodium [43]. In contrast, membrane ruffling occurs when no or not stable enough adhesions can be formed during force appli-cation on the lamellipodium leading to detachment of the cell from the substrate [43].
2.3.2 Details of Spreading in Platelets
When a platelet spreads on a surface, its discoid shape is first converted into a rounded/spheroid shape [44]. Further spreading includes filopodia or is con-ducted simply with lamellipodia [2]. The authors (Allenet al. [2]) used the termini pseudopodia and hyalomer, respectively. However, from the description they gave and the images they provided hyalomers are most likely lamellipodia and pseu-dopodia are most likely filopodia. They also stated that hyalomers resemble lamel-lipodia in their dynamics [2]. Thus, the terms filopodia and lamellamel-lipodia will be used in the following instead of pseudopodia and hyalomer.
Different types of spreading have been described for platelets seeded on sili-conized glass at 29◦C [2]. For platelets spreading via contribution of filopodia, the filopodia extend before the onset of lamellipodial spreading in between the filopo-dia [2]. The lamellipodium can also extend laterally from an extended filopodium [2]. Spreading via lamellipodia is achieved by outwards spreading of these lamel-lipodia which either occurs symmetrically or asymmetrically [2]. Filopodia are not needed for spreading via lamellipodia [2]. Furthermore, a single platelet can display more than one of these types of spreading in different regions of its pe-riphery [2]. Platelet spreading on siliconized glass is accomplished within as little as 10−12 minutes and seldomly takes more than 30 minutes [2]. On smooth glass coverslips covered with fibrinogen, filopodia are observed to develop spatially isotropic for murine platelets [56]. Spreading is achieved via polymerization of
14
Molecular Details and Mechanisms of Spreading 2.3 actin filaments [44, 100] which is driven by either uncapping of barbed ends of
ex-isting actin filaments [44] or by nucleation via Arp2/3 [36]. In activated platelets 60−85 % of the total actin content is integrated into the cytoskeleton [17]. The role of the wrinkled plasma membrane of quiescent platelets and of the OCS in spreading has been detailed above (section 2.1.3).
During spreading the platelet becomes flat and granules as well as organelles are translocated to the cell center [44]. The extended lamellipodia can then ruffle and retract [2, 44] and filopodia emerge from the center of the cell [44]. Lamellipodia are filled with a dense network consisting of actin filaments that are about 0.5µm long [44] and in a completely spread platelet the lamellipodium is only 50−100 nm thick [2]. Filopodia consist of bundles of long actin filaments [44] and contain microtubuli [44, 73].
Park et al. [72] examined platelet area over time for spreading on fibrinogen-coated glass at room temperature. In this study, platelets reach an area of about 50µm2after approximately 1 hour of spreading [72]. Additionally, they examined platelet circularity and showed that at low fibrinogen concentrations of smaller than 0.16µg/cm2 circularity depends on fibrinogen concentration whereas for higher concentrations the area of platelets is elevated and the circularity stays constant [72]. Lee et al. [56] investigated spreading of murine blood platelets on fibrinogen coated glass coverslips. This spreading shows a fast first, isotropic spreading phase that lasts about 2 minutes [56]. Thereafter, spreading slows down and is thought to be accompanied by formation of stable adhesions [56].
PAR4 is a thrombin receptor [11]. If this thrombin receptor is activated, platelets start spreading nearly instantaneously upon contact with the surface and filopo-dia only persist for 4 minutes after the onset of spreading while lamellipofilopo-dia form during the whole spreading process [56]. These findings indicate a spreading via lamellipodia upon activation of PAR4 [56]. The onset of lamellipodia forma-tion depends on the amount of fibrinogen on the substrate with more fibrinogen (100µg/ml compared to 1µg/ml) leading to faster lamellipodia formation [56].
Without activation of PAR4 the number of filopodia is increased to about 5 com-pared to about 2 with receptor activation [56]. The duration of filopodia formation is increased if PAR4 is not activated and the filopodia number reaches a plateau after about 4 minutes [56]. Spreading speed seems to be neither influenced by the protein coating of coverslips nor by activation of PAR4 [56]. However, the shape changes of platelets seem to be influenced by fibrinogen, since additional fibrinogen coating on dimethyldichlorosilane coated glass leads to decreases in
Chapter 2 STATE OF THE ART
circularity which are not seen without fibrinogen coating [72].
While platelets spread on fibrinogen coated surfaces, they can interact with the fib-rinogen and redistribute it with thrombin accelerating this redistribution [37]. It was observed by Feuersteinet al. [37] that platelets shrink after they have reached their maximal area and that the regions of the redistributed fibrinogen have a size resembling the maximal platelet area.
Newly arrived platelets which only attach to other already spread platelets but not the underlying substrate, i.e. coverslip, extend filopodia until these filopodia come in contact with the substrate and subsequently start to spread on the substrate [2].
The lamellipodia of neighboring platelets in contact with the substrate often show an overlap but spreading is not hindered [2].