End binding protein 1 (EB1) binds +end of MTs, localizing to the distal tips of MTs, the centrosome or MT ends in the mitotic spindle. It is sometimes referred to as the „master regulator of plus-end-tracking proteins (+TIPs)” for its ability to recruit various +TIPs and thus influence not only MTs themselves, but other processes such as membrane-anchoring or actin polymerization as well (36). EB1 binding to MTs stimulates MT elongation, whereas its dissociation causes slower growth of MTs and can influence cells’ direction of movement (37). Interestingly, depletion of EB1 has much bigger consequences on migration and protrusion branching in 3D migration. Whereas in 2D there is no significant effect on protrusions and overall migration speed, in 3D, depleting EB1 in mesenchymally migrating cells results in slower invasion and defects in cell directionality (38). In mesenchymal cells, EB1 mediates the binding of proteins CLASP1 and SLAIN2 to MTs, which contributes to their growth persistence in protrusions by reducing catastrophes ( Figure 2 ). This was shown to be necessary for the invasive shape and 3D mesenchymal invasion (39).

IQ motif-containing GTPase-activating protein 1/2/3 (IQGAP1-3) are scaffold proteins that integrate many signaling pathways via direct and indirect binding of over 90 proteins (40), including RhoGTPases ( Figure 1 ).

IQGAP proteins directly affects the dynamics of both the actin and the microtubule cytoskeleton. IQGAP1 is able to cross-link F-actin filaments (41–44) or by binding barbed ends of actin filaments, inhibit their growth, and protect them from depolymerization (42). IQGAP1 interacts with N-WASP and Arp2/3 forming a complex able to nucleate branched actin filaments (45, 46). In agreement, IQGAP1 was shown to be localized at the leading edges of polarized cells and in the lamellipodia of motile cells (43, 44, 46–48). One of the main functions of IQGAP1 is anchoring MTs to the cell cortex enabling directional movement.

With regard to MTs in migration, one of the main functions of IQGAP1 is anchoring MTs to the cell cortex enabling directional movement, a process regulated by multiple mechanisms. IQGAP1 interacts with +TIP CLIP-170 and activated Rac1 and Cdc42 (but not RhoA) forming a tripartite complex leading MTs to the cortex to areas with activated Rac1/Cdc42. Impaired binding of IQGAP1 caused by mutation at its C-terminus results in multiple leading edges in cells (49, 50). In addition, IQGAP1 and active Rac1/Cdc42 form a complex with adenomatous polyposis coli (APC) cortical filaments and depletion of either APC or IQGAP1 inhibits polarized migration (51). Another +TIP linking IQGAP1 to MTs at the cell cortex is protein SKAP (+TIP known to bind to EB1), yet again, disrupting the interaction of SKAP and IQGAP1 impairs cell migration (52).

Overall, IQGAP1 overexpression can promote cell migration and neurite outgrowth, while its depletion leads to decreased cell migration (47, 53, 54), providing evidence that the actin/microtubule crosstalk is necessary for polarized, directed cell movement.

Navigator proteins 1-3 (NAV1/2/3) are microtubule + end binding proteins that are implicated in axon guidance and neurite outgrowth in the brain (55, 56). In neurons, NAV1 is localized at the neurite tips where it binds actin-rich domains and crosslinks with the MTs in an EB1-dependant manner (55, 57). It also forms a complex with the protein TRIO, a guanine nucleotide exchange factor (GEF) known to activate Rac1 and RhoG (56–59). Thus, NAV1 does not influence MTs polymerization per se, but it promotes +end rescue and prevents catastrophes in the F-rich-domain periphery. Cells depleted in NAV1 were shown to be defective in migration during embryonic development (55). Similarly, NAV3 binds to MTs +end via EB1 and increases their polarized growth in response to EGF signaling in cancer cells by protecting them from catastrophes. Cells overexpressing NAV3 displayed higher MTs acetylation and resistance to nocodazole treatment, both distinctive of stabilized MTs (60). On the other hand, cells depleted in NAV3 showed more random migration and lost the ability to migrate persistently after EGF induction (60). Since persistent, protrusion dependent migration is characteristic of mesenchymal cells, Navigators are likely to be more crucial for mesenchymal than amoeboid migration.

Stathmin, also known as oncoprotein 18 (Opt18), is an important microtubule destabilizing protein able to cause MT depolymerization. Two modes of action have been described – first, by stimulating MT catastrophes (61), and second, by sequestrating αβ tubulin dimers to prevent their assembly (62) - which seems to depend on environmental conditions (63).

Stathmin is regulated by several kinases, and its phosphorylation on at least one of its four serins suppresses its destabilizing activity (64, 65). Moreover, Stat3 can bind to stathmin at its tubulin-binding site to prevent its function (66). Similarly, p27Kip (see further) binding prevents stathmin-regulated MT destabilization (67, 68). Of note, p27Kip expression is partially regulated by Stat3 (69).

The regulation of stathmin phosphorylation is key for cell migration, as it enables creation of a gradient of MT stability from the leading edge to the trailing edge. At the leading edge, high levels of Cdc42 and Rac1 prevent stathmin activation (27) resulting in stabilized MTs in these parts of the cell. On the trailing edge stathmin is not phosphorylated by Cdc42 or Rac and its MTs-destabilizing activity increases, leading to MT network disassembly and proper contraction of this end during cell movement (70) ( Figure 1 ).

Generally, stathmin is a strong pro-migratory factor as evidenced by a number of studies (67, 71), although in in certain conditions its inhibition may stimulate migration (66). This may be dependent on whether the cells utilize the mesenchymal and amoeboid type of invasion, since stathmin SQ18E, which is unable to undergo inhibitory phosphorylation, was directly linked to promotion of the round, amoeboid-like phenotype in sarcoma cells (71) ( Figure 2 ).

Protein p27^Kip is mainly known for its nuclear role as a cyclin dependent kinase inhibitor and thus inhibitor of cell cycle progression (72). However, it also plays an important role in the cytoplasm where it interacts with other proteins through its C-terminal domain to modulate cell motility and tumor progression (67, 73, 74). One of the interaction partners of p27^Kip is stathmin, binding of which inhibits stathmin´s activity leading to more stable MTs. In mesenchymal cells, this interaction limits migratory potential (67, 68) ( Figure 1 ).

P27^Kip does not influence only the MT network, it also affects actin filament reorganization. It was shown that p27^Kip is able to interact with RhoA, inhibiting its ability to bind GEFs and thus preventing its activation (73). Cytoplasmatic p27^Kip affects the actin cytoskeleton also indirectly via Rac1 dependent actin rearrangement and polymerization, leading to an increase in cell migration (75).

Of note, transformed fibroblasts lacking p27 ^Kip adopted a rounded morphology with cortical actin formation and loss of β1 integrin clusters, corresponding to the mesenchymal-amoeboid transition (76). Here both the actin and MT cytoskeleton were altered, showing a synergic effect of p27 ^Kip in regulation of cell invasion. In macrophages, p27 ^Kip contributes to the onset of mesenchymal migration by inhibition of RhoA/ROCK and lack of p27 ^Kip promotes amoeboid migration (77). Collectively, cytoplasmic p27 regulates invasion plasticity and exerts pro-mesenchymal signaling ( Figure 2 ).

mDia2 protein (mammalian homolog of Drosophila diaphanous; also known as DIAPH3) belongs to the group of formins (78), which are proteins involved in actin nucleation and elongation. In addition, mDia2 is able to bind and stabilize MT polymers. Its silencing leads to MT catastrophes and rewires EGFR and ERK signaling, resulting in increased ameboid traits (79). A different study showed that mDia2 in complex with its inhibitor induces amoeboid morphology in cells (80). Accordingly, depletion of mDia2 promoted individual dissemination of amoeboid cells from tumor spheres (81). Importantly, this affects cancer treatment, since cells without mDia2, exerting unstable MTs and amoeboid characteristics, are more susceptible to taxane chemotherapy (82). Overall, mDia2 regulates invasion plasticity through MT stabilization and actin nucleation, and its absence promotes the amoeboid invasion phenotype ( Figure 2 ), suggesting its pro-mesenchymal role.

An important molecule which interconnects MT dynamics and the actomyosin network is guanosine exchange factor H1 for GTPase RhoA (GEFH1), also known as ARHGEF2. GEFH1 is a one of the few GEFs that associate with polymerized microtubules. Upon MT depolymerization, GEFH1 is released and its guanosine exchange activity increases, leading to higher activation of RhoA and its downstream signaling ( Figure 1 ). This mechanism regulates RhoA activity in different parts of the migrating cell based on MT dynamics (83). Of note, GEFH1 activity can be further potentiated by phosphorylation mediated by Src kinase at the protruding cell edge (84). GEFH1 also affects focal adhesions (FAs) turnover in migrating cells and dysfunctional GEFH1-RhoA activation can disrupt cell motility (83). In lymphoma cells, Stat3 signaling induced destabilization of MTs, which led to the release of GEFH1 and as a result, amoeboid invasion (85).

In summary, the GEFH1-RhoA signaling pathway is induced by MTs destabilization, and its extent promotes either mesenchymal or amoeboid migration. In mesenchymal cells, activation of this pathway by dynamic growth of MTs contributes to protrusion regulation and faster FA turnover. In cells with disrupted MTs, GEFH1-RhoA signaling dominates and induces amoeboid invasion through the RhoA/ROCK pathway ( Figure 2 ).

There is also a large group of microtubule associated proteins (MAPs) that bind to MTs, but surprisingly, despite their unified function of stabilizing MT, they have different roles in tumor progression and may function as both pro- and anti-metastatic factors (86–88). We hypothesize that this inconsistency may be due to the different dependency of the amoeboid and mesenchymal migration on the MT network. Specifically, a change in invasive behavior was connected to MAP1B, MAP2, MAP4 and MAP7. Although there is no direct link between invasion plasticity and MAPs described so far, they likely participate by indirect signaling affecting RhoGTPases signaling. For example, MAP1B increases Rac1 activity through interaction with TIAM-1, a Rac1-GEF (89). Another possible mechanism involves phosphorylation of MAP4, which inhibits its MT stabilizing activity (90) and leads to MT disruption, which can increase RhoA through GEHH1 release (91).

For efficient cell migration, cells need to be polarized to maintain directionality of movement. The polarity of the cell is determined, amongst others, by the position of the nucleus and centrosome, forming the nuclear-centrosomal axis. By moving the centrosome, also known as the microtubule organizing center (MTOC), to a different position within the cell, the polarity of a cell can be shifted (92).

When cells gain a mesenchymal migratory phenotype, the centrosome position shifts to a central position in front of the nucleus relative to the future movement of the cell (93). However, this positioning of the centrosome is not a rule, as some studies show localization of the centrosome behind the nucleus (94, 95). Some studies also show that the position of the centrosome is influenced by the geometrical limitation of the cell’s surroundings rather than its function (96).

In most amoeboid cells, the location of the MTOC corresponds to mesenchymal cells. Interestingly though, in amoeboid leukocytes, the MTOC is placed behind the nucleus toward the cell rear ( Figure 2 ) (97, 98) and participates in the path finding mechanism. Once the cell’s nucleus, which represents the bulkiest part of the cell, and the associated MTOC successfully pass through a pore, protrusions directed to the smaller pores are retracted and the cell continues its movement through the path of least resistance. In this case, disrupting MTs leads to loss of cell coherency and results in fragmentation of the cell (97).

FAs are integrin-based structures responsible for strong adhesion of cells to the ECM. Importantly, they also transmit information about the surrounding environment such as its stiffness by signaling to cytoskeleton associated proteins (102–104). Both Rac and RhoA play role in FA regulation – whereas Rac activity is prominent in the formation of FAs, RhoA activity and Rac inhibition are needed for the maturation of FAs (105, 106).

The main role of MTs within FAs is the transport of integrins and metalloproteases (MMPs) to the cell membrane and the regulation of FAs turnover (107). Although there is no evidence of direct binding of MTs to FAs so far, MTs are known to be guided towards them and anchored in their proximity (108–111). The targeting of mature FAs by MTs results in FA disassembly and cell edge retraction, and preventing the contact between MTs and FAs leads to enlarged FAs (112). In agreement, nocodazole-induced MTs depolymerization also results in larger FAs, whereas MT regrowth after nocodazole washout disassembles FAs (112–114). Nevertheless, these studies were done in 2D environments, where the structure, composition and dynamics of FAs is different than in 3D environments (115), and many mechanisms valid in 2D systems are yet to be verified for 3D migration.

Podosomes and invadopodia are similar actin-based structures that play role in cell migration and ECM degradation, which is a key feature of mesenchymal invasion. Whereas podosomes are small, dot-like dynamic structures at the leading edge or organized rings exhibiting shallow ECM degradation, invadopodia are larger, irregularly shaped clusters usually localized in the central area of the cell. They are less dynamic and form outstretched extensions into the matrix resulting in deeper and more focused ECM degradation (116).

An intact MT network is necessary for the formation of podosomes since MT depolymerization (induced e.g. by nocodazole) leads to podosomal disassembly (117, 118). MTs are directed to podosomes through +TIPs such as EB1 and CLASPs (119, 120). Targeting of podosomes by MTs is associated with their higher dynamics, and podosomes without MTs show increased stability similarly to FAs (109, 112, 121).

The lifespan of MTs in podosomal structures is regulated by RhoGTPases. Inhibition of RhoA is able to increase the stability of MTs in podosomes, promoting podosome belt assembly (122). Accordingly, activation of RhoA leads to podosome disassembly through the RhoA/ROCK/MLCP axis as actomyosin contractility increases podosomes turnover and their disassembly (123–125). Altering Cdc42 and Rac1 activity was also shown to disrupt podosomes (118, 122, 126).

Invadopodia are invasive structures commonly found in various cancers (127). Unlike podosomes, microtubules are not required for invadopodia formation but are necessary for their elongation and correct function (128, 129). Invadopodia first form as a smaller actin-based structure with microtubules excluded from their core (130). Only after maturation of invadopodia, intermediate filaments and microtubules (typically 1-2 MTs per protrusion) invade their structure and allow invadopodia elongation. The microtubules that invade the shaft of invadopodia are stable, whereas at the base of the invadopodium more dynamic MTs are found (128). Accordingly, MT depolymerization does not affect the formation of small invadopodia, but limits their elongation and maturation (129).

One important role of MTs in invadopodia is the polarized trafficking of components such as matrix MMPs to the cell membrane (128, 131, 132). Moreover, the MT associated protein IQGAP1 accumulates in invadopodia where it interacts with exocyst components, and this interaction promotes invadopodia proteolysis by accumulation of MT1-MMP in a MT-regulated manner (133). Also, the exocytosis of MMP2 and MMP9 in melanoma cells is MTs-dependent (132) supporting the role of MTs in trafficking invadopodia components.

Microtubule drugs, due to their immense effect on MT structure and dynamics, have extensive impact on cell behavior. They are commonly used as chemotherapeutic agents based on their ability to arrest cell proliferation and cause cell death (147). Nevertheless, MT drugs exhibit more extensive behavior than just antimitotic effects, including deregulation of cell migration (148). They also interfere with invasion plasticity manifesting different effects on each invasion mode. Drugs inhibiting MT polymerization promote the amoeboid mode as it is less MT-dependent ( Figure 2 ). For example, vincristine which is able to sequester tubulin dimers and prevent MT polymerization, induces the amoeboid phenotype through GEFH1/RhoA signaling (149). Similar results were observed with nocodazole, which also leads to MT disassembly and subsequent RhoA activation (150). In fibroblasts, nocodazole treatment prevented cells to adopt an elongated morphology and instead induced a round morphology, typical of the amoeboid phenotype (151). Treatment of cells with paclitaxel (taxol) stabilizes microtubules, but does not promote mesenchymal migration, instead it induces a non-motile phenotype (152–154). In lymphocytes that utilize the amoeboid migration mode, taxol treatment inhibited migration in both 2D and microstructured environments, whereas nocodazole treatment increased membrane blebbing without increase in migration in 2D (155), but was able to promote amoeboid invasion in 3D (156).

The wide clinical usage of classic MT drugs is hampered by notable drug resistance and toxicity, fueling the chase for novel compounds with improved characteristics (157), many of which are in clinical trials (158). Moreover, in recent years it has become evident that the requirement for anti-metastatic behavior should be evaluated as well as primary growth shrinkage (159–162). In point of fact, the prototypic antimitotic drugs paclitaxel and vincristine can in certain instances promote metastasis (163, 164). On the other hand, vinorelbine treatment in mice reduced metastasis more effectively than primary tumor growth (165). Another microtubule inhibitor, eribulin, exerts migrastatic behavior both in experimental conditions (166, 167) and in patients with advanced metastatic breast cancer (168).

Taken together, a suitable combination of microtubule drugs with invasion specific inhibitors could synergically target cell proliferation and both amoeboid and mesenchymal invasion, i.e., elicit both anti-proliferative and migrastatic effects.