Localization of Actin mRNA during the Establishment of Cell Polarity and Early Cell Divisions in Fucus Embryos

Localization of mRNA is a well-described mechanism to account for the asymmetric distribution of proteins in polarized somatic cells and embryos of animals. In zygotes of the brown alga Fucus, F-actin is localized at the site of polar growth and accumulates at the cell plates of the first two divisions of the embryo. We used a nonradioactive, whole-mount in situ hybridization protocol to show the pattern of actin mRNA localization. Until the first cell division, the pattern of actin mRNA localization is identical to that of total poly(A)+ RNA, that is, a symmetrical distribution in the zygote followed by an actin-dependent accumulation at the thallus pole at the time of polar axis fixation. At the end of the first division, actin mRNA specifically is redistributed from the thallus pole to the cell plates of the first two divisions in the rhizoid. This specific pattern of localization in the zygote and embryo involves the redistribution of previously synthesized actin mRNA. The initial asymmetry of actin mRNA at the thallus pole of the zygote requires polar axis fixation and microfilaments but not microtubules, cell division, or polar growth. However, redistribution of actin mRNA from the thallus pole to the first cell plate is insensitive to cytoskeletal inhibitors but is dependent on cell plate formation. The F-actin that accumulates at the rhizoid tip is not accompanied by the localization of actin mRNA. However, maintenance of an accumulation of actin protein at the cell plates of the rhizoid could be explained, at least partially, by a mechanism involving localization of actin mRNA at these sites. The pattern and requirements for actin mRNA localization in the Fucus embryo may be relevant to polarization of the embryo and asymmetric cell divisions in higher plants as well as in other tip-growing plant cells.     

Localization of actin networks during early development of Tubifex embryos

In precleavage zygotes of Tubifex, actin filaments segregate to the animal and vegetal poles forming the polar actin filament networks (AFNs). In this study, the fate of the polar AFNs during early development of Tubifex embryos has been followed using rhodamine-phalloidin as a specific stain for F-actin. During the first two cleavages, which are unequal and meridional, the polar AFNs are retained at the regions of cells corresponding to the poles of the precleavage zygote; thereby, they are segregated to the CD-cell at the 2-cell stage then to the D-cell at the 4-cell stage. As the mitotic apparatus forms in the D-cell, however, the vegetal polar AFN translocates toward the animal pole of the cell where the mitotic apparatus is located and unites with the animal polar AFN there. This redistribution of the AFNs is impaired by colchicine treatment, suggesting the involvement of microtubules. Thereafter, the unified AFN is found to be associated with nuclear regions of the macromeres of the D-cell line, and finally partitioned to the teloblast precursors 2d and 4d and an endodermal cell 4D. Cytochalasin B experiments indicate that the AFNs play a cytoskeletal role in generating and maintaining the spatial organization of the cytoplasm which gives rise to the intracellular localization of the cytoplasm and the mitotic apparatus orientations. The developmental and cellular significance of the AFNs is discussed in relation to the localization of developmental potential and the regulation of the mitotic apparatus organization in the Tubifex embryo.     

Establishment of cell polarity during early plant development

Cellular asymmetries have been proposed to play a role in plant embryogenesis. Genetic studies of Arabidopsis and other experimental approaches in several plant species have addressed the origins of cellular asymmetry in specific cases. Although zygote polarity, which precedes the formation of the apical—basal axis of the embryo, is normally aligned with that of the surrounding maternal tissue, isolated single somatic cells that give rise to embryos in culture appear to become polar in the absence of maternal factors. Gene expression patterns reveal the developmental consequences of cellular asymmetries occurring at later stages of embryogenesis. Genetic evidence suggests that these cellular asymmetries are established in response to as yet unidentified signals from adjacent cells.     

Ca2+ and Calmodulin Dynamics during Photopolarization in Fucus serratus Zygotes

The role of Ca2+ in zygote polarization in fucoid algae (Fucus, Ascophyllum, and Pelvetia species) zygote polarization is controversial. Using a local source of Fucus serratus, we established that zygotes form a polar axis relative to unilateral light (photopolarization) between 8 and 14 h after fertilization (AF), and become committed to this polarity at approximately 15 to 18 h AF. We investigated the role of Ca2+, calmodulin, and actin during photopolarization by simultaneously exposing F. serratus zygotes to polarizing light and various inhibitors. Neither removal of Ca2+ from the culture medium or high concentrations of EGTA and LaCl3 had any effect on photopolarization. Bepridil, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester, nifedipine, and verapamil, all of which block intracellular Ca2 release, reduced photopolarization from 75 to 30%. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-L-naphthalenesulfonamide and trifluoperazine inhibited photopolarization in all zygotes, whereas N-(6-aminohexyl)-L-naphthalenesulfonamide had no effect. Cytochalasin B, cytochalasin D, and latrunculin B, all of which inhibit actin polymerization, had no effect on photopolarization, but arrested polar axis fixation. The role of calmodulin during polarization was investigated further. Calmodulin mRNA from the closely related brown alga Macrocystis pyrifera was cloned and the protein was expressed in bacteria. Photopolarization was enhanced following microinjections of this recombinant calmodulin into developing zygotes. Confocal imaging of fluorescein isothiocyanate-labeled recombinant calmodulin in photopolarized zygotes showed a homogenous signal distribution at 13 h AF, which localized to the presumptive rhizoid site at 15 h AF.     

RAC1 regulates actin arrays during polarity establishment in the brown alga, Silvetia compressa

Multicellular development has evolved independently on numerous occasions and there is great interest in the developmental mechanisms utilized by each of the divergent lineages. Fucoid algae, in the stramenopile lineage (distinct from metazoans, fungi and green plants) have long been used as a model for early development based on unique life cycle characteristics. The initially symmetric fucoid zygote generates a developmental axis that determines not only the site of growth, but also the orientation of the first cell division, whose products have distinct developmental fates. Establishment and maintenance of this growth axis is dependent on formation of a filamentous actin array that directs vesicular movement, depositing new membrane and wall material for development of the rhizoid. What is not well known, is how formation and placement of the actin array is regulated in fucoid algae. A candidate for this function is Rac1, a small GTPase of the highly conserved Rho family, which has been implicated in controlling the formation of actin arrays in diverse eukaryotes. We demonstrate that Rac1 is not only present during formation of the filamentous actin array, but that its localization overlaps with the array in polarizing zygotes. Pharmacologically inhibiting Rac1 activity was shown to impede formation and maintenance of the actin array, and ultimately polar growth. Evidence is provided that a requirement of Rac1 function is its ability to associate with membranes via a post-translationally added lipid tail. Taken together, the data indicate that Rac1 is a necessary participant in establishment of the growth pole, presumably by regulating the placement and formation of the actin array. A role for Rac1 and related proteins in regulating actin is shared by animals, plants, fungi and with this work, brown algae, thus a conserved mechanism for generating polarity is in operation in unique eukaryotic lineages.     

Separation of processes associated with differentiation of two-celled Fucus embryos

Various inhibitors were used to separate the overlapping processes of polar axis fixation, intracellular localizations forming a polar cell, and cell division, all of which are essential for cellular differentiation in two-celled embryos of Fucus distichus L. Powell. Cycloheximide and sucrose delayed the appearance of a polar cell (rhizoid formation) without inhibiting the fixation of a polar axis. Cytochalasin B, at 10 μg/ml, reversibly inhibited rhizoid formation without altering cell division. At higher concentrations (50–100 μg/ml) given in short pulses, cytochalasin affected the orientation and delayed the fixation of a light-induced polar axis with no qualitative effect on cell division. Disruption of the mitotic apparatus and prevention of cell division by colchicine had no influence on rhizoid formation or on the photopolarization of the developmental axis.     

Overexpression of an Arabidopsis formin stimulates supernumerary actin cable formation from pollen tube cell membrane

Formins, actin-nucleating proteins that stimulate the de novo polymerization of actin filaments, are important for diverse cellular and developmental processes, especially those dependent on polarity establishment. A subset of plant formins, referred to as group I, is distinct from formins from other species in having evolved a unique N-terminal structure with a signal peptide, a Pro-rich, potentially glycosylated extracellular domain, and a transmembrane domain. We show here that overexpression of the Arabidopsis formin AFH1 in pollen tubes induces the formation of arrays of actin cables that project into the cytoplasm from the cell membrane and that its N-terminal structure targets AFH1 to the cell membrane. Pollen tube elongation is a polar cell growth process dependent on an active and tightly regulated actin cytoskeleton. Slight increases in AFH1 stimulate growth, but its overexpression induces tube broadening, growth depolarization, and growth arrest in transformed pollen tubes. These results suggest that AFH1-regulated actin polymerization is important for the polar pollen cell growth process. Moreover, severe membrane deformation was observed in the apical region of tip-expanded, AFH1-overexpressing pollen tubes in which an abundance of AFH1-induced membrane-associated actin cables was evident. These observations suggest that regulated AFH1 activity at the cell surface is important for maintaining tip-focused cell membrane expansion for the polar extension of pollen tubes. The cell surface-located group-I formins may play the integrin-analogous role as mediators of external stimuli to the actin cytoskeleton, and AFH1 could be important for mediating extracellular signals from female tissues to elicit the proper pollen tube growth response during pollination.     

Polarity of the ascidian egg cortex before fertilization.

The unfertilized ascidian egg displays a visible polar organization along its animal-vegetal axis. In particular, the myoplasm, a mitochondria-rich subcortical domain inherited by the blastomeres that differentiate into muscle cells, is mainly situated in the vegetal hemisphere. We show that, in the unfertilized egg, this vegetal domain is enriched in actin and microfilaments and excludes microtubules. This polar distribution of microfilaments and microtubules persists in isolated cortices prepared by shearing eggs attached to a polylysine-coated surface. The isolated cortex is further characterized by an elaborate network of tubules and sheets of endoplasmic reticulum (ER). This cortical ER network is tethered to the plasma membrane at discrete sites, is covered with ribosomes and contains a calsequestrin-like protein. Interestingly, this ER network is distributed in a polar fashion along the animal-vegetal axis of the egg: regions with a dense network consisting mainly of sheets or tightly knit tubes are present in the vegetal hemisphere only, whereas areas characterized by a sparse tubular ER network are uniquely found in the animal hemisphere region. The stability of the polar organization of the cortex was studied by perturbing the distribution of organelles in the egg and depolymerizing microfilaments and microtubules. The polar organization of the cortical ER network persists after treatment of eggs with nocodazole, but is disrupted by treatment with cytochalasin B. In addition, we show that centrifugal forces that displace the cytoplasmic organelles do not alter the appearance and polar organization of the isolated egg cortex. These findings taken together with our previous work suggest that the intrinsic polar distribution of cortical membranous and cytoskeletal components along the animal-vegetal axis of the egg are important for the spatial organization of calcium-dependent events and their developmental consequences.     

Confocal Microscopy Study of <Emphasis Type="Italic">Arabidopsis</Emphasis> Embryogenesis Using GFP:mTn

Embryogenesis in transgenic Arabidopsis plants with GFP:mTn, a chimeric fusion of soluble shifted green fluorescent protein and a mouse actin binding domain, was studied. Confocal laser scanning microscopy was used to determine patterns of formation and cellular responses during asymmetric cell division. Before such cells divide, the nucleus moves to the position where new cell walls are to be formed. The apical–basal axis of the embryo develops mainly at the zygote to octant stage, and these events are associated with asymmetric divisions of the zygote and hypophyseal cells. Formation of the radial axis is established from the dermatogen to the globular-stage embryo via tangential cell division within the upper tiers. Bilateral symmetry of the embryo primarily happens at the triangular stage through zig-zag cell divisions of initials of the cotyledonary primordia. All stages of embryogenesis are described in detail here.     

Bipolar segregation of mitochondria,actin network,and surface in the Tubifex egg: Role of cortical polarity

The role of the cortex in ooplasmic segregation of the yolky eggs of Tubifex has been studied by epifluorescence microscopy. Living eggs labeled with rhodamine 123 and fine carbon particles placed on the surface showed that, following the second polar body formation, the egg surface cosegregates with subcortical mitochondria in a bipolar fashion, viz. toward the animal and vegetal poles in the animal and vegetal hemispheres, respectively. The egg surface of each pole moves spirally while the equatorial surface appears to remain stationary during this process. The rhodamine-phalloidin staining of whole eggs reveals that actin networks cosegregate with mitochondria. Isolated cortices which were stained with rhodamine-phalloidin demonstrated that cortical actin is organized bipolarly and that, during ooplasmic segregation, it undergoes reorganization directed toward both poles of the egg. The cortical polarity expressed as actin organization is not disrupted by centrifugal force sufficient to stratify the egg cytoplasm into five layers. The surface of a centrifuged egg moves according to the original cortical polarity. This surface movement is accompanied by the reorganization of cortical actin which appears to be identical to that in intact eggs. Other centrifugation experiments have demonstrated that the connection of the subcortical cytoplasm to the cortex is resistant to a centrifugal force of up to 650g. The nature of cortical polarity and its role in ooplasmic segregation are discussed in the light of the present results.     

Actin nucleoskeleton in embryonic development and cellular differentiation

Dynamic assembly and disassembly of actin proteins play a key role in the cytoskeleton, but the cellular functions of actin are not only restricted to the cytoplasmic compartment. Recent studies have shown that actin spatiotemporally changes its polymerized state in the nucleus as well and such dynamic nature of actin is relevant to key nuclear events including gene expression, DNA damage response and chromatin organization. In this review, we highlight emerging roles of actin in the nuclear compartment especially in the context of embryonic development and cellular differentiation. We first explain how the actin nucleoskeleton can be formed and function in cells. Notably, nuclear actin dynamics are greatly altered when cell fates change, such as after fertilization and T cell differentiation. We discuss how the dynamic actin nucleoskeleton contributes to accomplishing developmental programs.     

The Rac1 inhibitor,NSC23766, depolarizes adhesive secretion,endomembrane cycling,and tip growth in the fucoid alga, <Emphasis Type="Italic">Silvetia compressa</Emphasis>

Proper cell morphogenesis is dependent on the establishment and expression of cellular polarity. In the fucoid zygote, cell shape is critical for establishing the developmental pattern of the adult, and is achieved by guiding insertion of new membrane and wall to the rhizoid tip. Selection and growth of the appropriate tip site are accompanied by formation of dynamic actin arrays associated with the actin-nucleating Arp2/3 complex. In eukaryotes, a major pathway for activation of the Arp2/3 complex is via the Rho family GTPase, Rac1, which stimulates the Scar/WAVE complex. To determine whether Rac1 controls actin nucleation in Silvetia compressa (J. Agardh) E. Serrao, T. O. Cho, S. M. Boo et Brawley, we tested the effects of the Rac1-specific inhibitory compound, NSC23766, on actin dependent processes and on actin arrays. We found that NSC23766 disrupted polar secretion of adhesive, polarization of endomembranes, and tip-focused growth in the rhizoid. Similarly, NSC23766 altered actin and Arp2 localization in the growing rhizoid. In contrast, NSC23766 had no effect on selection of the growth site or on cytokinesis. These data suggest that Rac1 participates in nucleation of specific actin arrays in the developing zygote.     

Morphological changes in the oocyte and its surrounding vestments during in vivo fertilization in the dasyurid marsupial Sminthopsis crassicaudata

This light and transmission electron microscopical study shows that the first polar body is given off before ovulation and that part of its cell membrane and that of the surrounding oocyte have long microvilli at the time of its ejection. Several layers of cumulus cells initially surround the secondary oocyte and first polar body, but the ovulated oocytes in the oviducts in the process of being fertilized do not have cumulus cells around them. Partly expelled second polar bodies occur in the oviduct; they are elongated structures that lack organelles and have electron-dense nuclei. A small fertilization cone appears to form around the sperm tail at the time of sperm entry into the egg and an incorporation cone develops around the sperm head in the egg cytoplasm. In three fertilized eggs a small hole was seen in the zona, which was presumably formed by the spermatozoon during penetration. Cortical granules, present in ovarian oocytes, are not seen in fertilized tubal or uterine eggs; release of their contents probably reduces the chances of polyspermy, although at least one polyspermic fertilized egg was seen and several other fertilized eggs had spermatozoa within the zona pellucida. In the zygote the pronuclei come to lie close together, but there was no evidence of fusion. A "yolk mass," which becomes eccentric before ovulation, is extruded by the time the two-cell embryos are formed, but many vacuoles remain in the non-yolky pole of the egg. A shell membrane of variable thickness is present around all uterine eggs but its origin remains undetermined.     

Surface alterations of fertilized surf clam (Spisula solidissima) eggs induced by concanavalin A

Fertilized Spisula eggs, incubated in ConA, were examined at periodic intervals to determine the effects of lectin binding on events of fertilization and cleavage. ConA was localized to specific regions of the vitelline layer and plasma membrane by reacting lectin-treated eggs with horseradish peroxidase and diaminobenzidine. In contrast to eggs, little reaction product was associated with the plasma membrane of spermatozoa. Sperm that fused with ConA-treated eggs failed to move into the cortex of the ovum and were observed as bulbous appendages at the surface of the zygote. Reorganization of sperm nuclei was inhibited, and male pronuclei failed to develop. ConA also inhibited polar body formation and cleavage. The maternally derived chromatin underwent meiosis, and the chromosomes normally taken into the first and second polar bodies were retained within the zygote. All of the maternally derived chromatin was organized within four or more female pronuclei which subsequently entered mitosis. The effects of ConA binding on events at the surface of fertilized Spisula eggs were abrogated by α-methyl-d-mannoside; succinyl-ConA only partially inhibited fertilization-related processes. The effects of ConA are discussed in terms of possible cross-linking of surface components of fertilized Spisula eggs which may inhibit deformation of the zygote cortex.     

Confocal and video imaging of cytoskeleton dynamics in the leech zygote

Ooplasmic segregation in the late interphase zygote of the leech Theromyzon trizonare is accomplished by reorganization of an ectoplasmic cytoskeleton formed by polar rings and meridional bands. The dynamic properties of this cytoskeleton were explored by time-lapse confocal and video microscopy. Cytoskeleton assembly was investigated in zygotes pulse-labeled with microinjected fluorophore-tagged or biotin-tagged dimeric tubulin and G-actin. Cytoskeleton disassembly was studied by comparing the linear dimensions of the cytoskeleton at different time points during late interphase. The relative distributions of F- and-G-actin were determined after microinjection of rhodamine-labeled actin and fluorescein-labeled DNase I. Results showed that labeled precursors were readily incorporated into a network of microtubules or actin filaments. Bipolar translocation of the rings and meridional bands was accompanied by the rapid assembly and disassembly of microtubules and actin filaments. Because labeled microtubules and microfilaments gradually decreased, the rate of cytoskeleton disassembly was greater than the rate of cytoskeleton assembly. Hence, ooplasmic segregation was accompanied by the rapid turnover of cytoskeletal components. Co-distribution of F- and-G-actin during mid and late interphase may favor polymer-monomer interchange. We conclude that cytoskeleton reorganization during foundation of cytoplasmic domains can be conveniently studied in the live leech zygote after microinjection of labeled precursors.     

Actin,more than just a housekeeping protein at the scene of fertilization

Since the first demonstration of sperm entry into the fertilized eggs of Mediterranean sea urchin Paracentrotus lividus by Hertwig (1876), enormous progress and insights have been made on this topic. However, the precise molecular mechanisms underlying fertilization are largely unknown. The two most dramatic changes taking place in the zygote immediately after fertilization are: (i) a sharp increase of intracellular Ca²⁺ that initiates at the sperm interaction site and traverses the egg cytoplasm as a wave, and (ii) the concomitant dynamic rearrangement of the actin cytoskeleton. Traditionally, this has been studied most extensively in the sea urchin eggs, but another echinoderm, starfish, whose eggs are much bigger and transparent, has facilitated experimental approaches using microinjection and fluorescent imaging methodologies. Thus in starfish, it has been shown that the sperm-induced Ca²⁺ increase in the fertilized egg can be recapitulated by several Ca²⁺-evoking second messengers, namely inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP), which may play distinct roles in the generation and propagation of the Ca²⁺ waves. Interestingly, it has also been found that the dynamic rearrangement of the actin cytoskeleton in the fertilized eggs plays pivotal roles in guiding monospermic sperm entry and in the fine modulation of the intracellular Ca²⁺ signaling. As it is well known that Ca²⁺ regulates the structure of the actin cytoskeleton, our finding that Ca²⁺ signaling can be reciprocally affected by the state of the actin cytoskeleton raises an intriguing possibility that actin and Ca²⁺ signaling may form a ‘positive feedback loop’ that accelerates the downstream events of fertilization. Perturbation of the cortical actin networks also inhibits cortical granules exocytosis. Polymerizing actin bundles also compose the ‘acrosome process,’ a tubular structure protruding from the head of fertilizing sperm. Hence, actin, which is one of the most strictly conserved proteins in eukaryotes, modulates almost all major aspects of fertilization.     

Intrinsic chiral properties of the Xenopus egg cortex: an early indicator of left-right asymmetry?

Vertebrate embryos define an anatomic plane of bilateral symmetry by establishing rudimentary anteroposterior and dorsoventral (DV) axes. A left-right (LR) axis also emerges, presaging eventual morphological asymmetries of the heart and other viscera. In the radially symmetric egg of Xenopus laevis, the earliest steps in DV axis determination are driven by microtubule-dependent localization of maternal components toward the prospective dorsal side. LR axis determination is linked in time to this DV-determining process, but the earliest steps are unclear. Significantly, no cytoskeletal polarization has been identified in early embryos capable of lateral displacement of maternal components. Cleaving Xenopus embryos and parthenogenetically activated eggs treated with 2,3-butanedione monoxime (BDM) undergo a dramatic large-scale torsion, with the cortex of the animal hemisphere shearing in an exclusively counterclockwise direction past the vegetal cortex. Long actin fibers develop in a shear zone paralleling the equator. Drug experiments indicate that the actin is not organized by microtubules, and depends on the reorganization of preexisting f-actin fibers rather than new actin polymerization. The invariant chirality of this drug response suggests a maternally inherited, microfilament-dependent organization within the egg cortex that could play an early role in LR axis determination during the first cell cycle. Consistent with this hypothesis, brief disruption of cortical actin during the first cell cycle randomizes the LR orientation of tadpole heart and gut.     

Automictic parthenogenesis of a geographically parthenogenetic mayfly,Ephoron shigae (Insecta: Ephemeroptera,Polymitarcyidae)

In the geographically parthenogenetic mayfly, Ephoron shigae, egg maturation and counts of chromosome number of unfertilized, parthenogenetic eggs were studied, in comparison with fertilized eggs from a bisexual population. The primary oocyte becomes mature through two successive maturation divisions. The first maturation division (meiotic division) takes place in the primary oocyte to produce a secondary oocyte and a first polar body. The second maturation division soon occurs in the secondary oocyte, in which the nucleus is divided into a mature egg nucleus (female pronucleus) and second polar body nuclei. The first polar body, in some cases, was successively divided into two polar bodies; in other instances, it was not divided. After the successive maturation division, the egg nucleus and the sister second polar body nucleus drew near to fuse into the zygote nucleus. The chromosome number was doubled in the zygote, and this conjugation initiates subsequent embryonic development. This suggests that, in E. shigae, the process of parthenogenetic recovery of diploidy is the automictic type categorized as the ‘terminal fusion’ pattern. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 335–343.     

Distribution and role of microfilaments during early events of sheep fertilization

The distribution of actin was studied during early events of sheep fertilization by fluorescence microscopy after staining with 7-nitrobenz-2-oxal-1.3 diazole (NBD)-phallacidin and anti-actin antibody and by electron microscopy after heavy meromyosin labelling. Unfertilized and fertilized eggs exhibited a continuous band of fluorescence with both NBD-phallacidin and anti-actin antibody. Unlike in mice, no high concentration of actin overlying the spindle was detected in ovulated sheep oocytes. At the site of sperm head incorporation, the fertilization cone developed above the decondensing male chromatin and was underlined by a submembranous area rich in microfilaments. A similar actin network was observed in the cortex of the second polar body. Cytochalasin D was used to investigate the role of actin during the fertilization process. This drug did not prevent sperm fusion and incorporation but inhibited polar body abstriction and fertilization cone development and retarded sperm tail incorporation. Moreover, in the presence of the drug, the anchorage of the metaphase II spindle at the surface of the egg was destroyed. The role of microfilaments in these early events is discussed.     

Contraction mechanisms in composite active actin networks

Simplified in vitro systems are ideally suited for studying the principle mechanisms of the contraction of cytoskeletal actin systems. To shed light on the dependence of the contraction mechanism on the nature of the crosslinking proteins, we study reconstituted in vitro active actin networks on different length scales ranging from the molecular organization to the macroscopic contraction. Distinct contraction mechanisms are observed in polar and apolar crosslinked active gels whereas composite active gels crosslinked in a polar and apolar fashion at the same time exhibit both mechanisms simultaneously. In polar active actin/fascin networks initially bundles are formed which are then rearranged. In contrast, apolar cortexillin-I crosslinked active gels are bundled only after reorganization of actin filaments by myosin-II motor filaments.