BIOLOGICAL OVERVIEW

Cdc42, a member of the Rho family of GTP binding proteins, functions in the formation of polarized actin structures, in elongation of cell shape, and in cell signaling. The functions of Cdc42 in developing tissues have been examined using loss-of-function mutations in Drosophila Cdc42. Cdc42 minus epithelial cells fail to elongate into a columnar cell shape and cannot maintain a monolayered epithelial structure. No requirement for Cdc42 in cell division or in activation of the Jun N-terminal kinase pathway is found. In addition, Cdc42 function is not required for cytoplasmic actin filament assembly in the nurse cells during oogenesis, although it may facilitate this process. Furthermore, the results indicate that Cdc42 plays a role in intercellular interactions between the germ line and the somatic follicle cells. These results confirm the role of Cdc42 in actin filament assembly and provide new insights into its functions in epithelial morphogenesis and regulating intercellular signaling events (Genova, 2000).

Members of the Ras superfamily of small GTPase proteins have been shown to act as molecular switches that regulate cell proliferation, cell fate, and a range of other cellular and developmental processes. Within this superfamily, the Rho family, including Rho, Rac, and Cdc42, has been particularly well studied because of its role in regulating cytoskeletal assembly and related cellular events such as membrane trafficking, cell polarity, cell adhesion, and cell elongation. More recently, these proteins have also been suggested to function as switches in signal transduction pathways, such as the Jun N-terminal kinase (JNK) pathway, with functions that extend beyond cytoskeletal regulation. In addition, numerous studies implicate Rho family members in processes associated with oncogenesis, either directly or as effectors of oncogenic pathways. Thus it appears that Rho family proteins play crucial roles in regulating cytoskeletal processes and a range of related cellular and developmental functions (Genova, 2000 and references therein).

Within the Rho family, the Cdc42 gene is unique in that it was originally identified as a cell cycle mutation in the yeast Saccharomyces cerevisiae. In the yeast, CDC42 function is necessary during the assembly of the bud site, an actin-rich structure that forms at one end of the budding yeast cell. Mutations in the yeast CDC42 gene disrupt the assembly of a ring of actin microfilaments that normally forms in the neck of the bud, thereby blocking bud formation and cell division. This phenotype has been interpreted to indicate that CDC42 mutations disrupt the ability of cells to construct localized or 'polarized' assemblies of actin microfilaments (Genova, 2000).

Experiments using cultured cells have implicated human Cdc42 in the formation of filopodia in growth factor stimulated cells, presumably via the assembly of membrane-associated microfilaments in these structures. Interestingly, other Rho family members appear to regulate the assembly of morphologically distinct cellular processes, such as lamellipodia and stress fibers. In addition, several lines of evidence implicate Cdc42 or another Rho family member as functioning upstream of the JNK pathway. Activated forms of both Rac and Cdc42 stimulate the JNK pathway in HeLa, NIH-3T3, and Cos cells. However, constitutively activated forms of Cdc42 and Rho appear to activate the JNK pathway in human kidney 293T cells. These seemingly contradictory results could indicate that the Rho family regulation of the JNK pathway occurs in a tissue-specific manner. Alternatively, it is possible that the mutationally activated forms of these highly related proteins result in nonspecific interactions between the structurally similar members of the Rho family (Genova, 2000).

The developmental functions of Cdc42 and the Rho family members have been investigated in Drosophila using dominant interfering or activating alleles. Dominant active and negative mutations in Rac disrupt axon outgrowth in the embryonic nervous system and affect myoblast fusion. Similar forms of Cdc42 affect neurite outgrowth in the peripheral nervous system but do not affect myoblast fusion. Dominant negative Cdc42 has also been shown to disrupt the process of dorsal closure late in embryonic development. Studies of Drosophila oogenesis using ectopically expressed dominant negative Cdc42 suggest that Cdc42 may function to regulate the assembly of actin microfilaments that are necessary for proper transfer of cytoplasm from the nurse cells to the oocyte. Dominant alleles of Cdc42 also affect the ability of imaginal epithelial cells to elongate into a columnar cell shape and disrupt the planar polarity (rather than the apical-basal polarity) of these epithelial sheets (Genova, 2000).

Genetic mutations in Drosophila Cdc42 (Fehon, 1997) were used to examine the effects of loss of Cdc42 function in developing tissues. Reduction of Cdc42 function in embryonic, imaginal, and follicular epithelial cells results in a loss of epithelial character -- mutant cells are unable to maintain a columnar cell shape, lose cohesion necessary to maintain cell sheets, and form multiple cell layers. Reduction of Cdc42 function in the embryonic epidermis leads to a failure in germ-band retraction and a degeneration of the ventral epidermis prior to the process of dorsal closure, yet axon outgrowth in the central and peripheral nervous systems still occurs. Functional Cdc42 is also required for proper differentiation of imaginal epithelial cells and in both the germ-line and the follicle cells for proper specification of the follicular stalk cells. In contrast, actin filament assembly in the developing nurse cells of the ovary is not dependent on Cdc42 function. In addition, contrary to predictions from previous studies, loss of Cdc42 function does not appear to affect activation of the JNK pathway during dorsal closure or cytokinesis in proliferating epithelial cells. Thus, these studies, which are the first to use loss-of-function Cdc42 alleles in a higher eukaryote, provide new insights into the functions of this ubiquitous cytoskeletal regulatory switch (Genova, 2000).

To determine the functions of Cdc42 during Drosophila development, the phenotypes of six previously isolated mutations in the Drosophila Cdc42 gene (Fehon, 1997) were examined. The molecular lesions associated with two of these alleles, Cdc42¹ and Cdc42², were previously characterized (Fehon, 1997). Of the lethal alleles, two (Cdc42¹ and Cdc42³) are missense mutations while the other (Cdc42⁴) is a mutation in the splice acceptor site (Genova, 2000).

Cdc42³ and Cdc42⁴ display a similar range of phenotypes and produce similar degrees of maternal-effect lethality, suggesting that they reduce Cdc42 function to similar levels. Rescue experiments using a Ubiquitin promoter-Cdc42¹ transgene indicate that Cdc42¹ is only partially rescuable, while all of the other alleles can be rescued completely, consistent with the notion that Cdc42¹ has dominant negative functions (Genova, 2000).

Previous studies of Cdc42 in yeast and mammalian cells have indicated that it plays a fundamental role in cellular morphogenesis. It is therefore surprising that lethal Cdc42 mutations in Drosophila display no embryonic phenotype (Fehon, 1997). One possible explanation for this apparent paradox is that Cdc42 is maternally expressed and loaded into the egg, thereby providing sufficient Cdc42 function for embryogenesis. Cdc42² germ-line clones could not be used to address this possibility because Cdc42 is required during oogenesis for the production of fertile eggs. Instead heteroallelic combinations of weak and strong alleles were used to produce females that, while viable, had reduced Cdc42 function. Examination of two such combinations, Cdc42³/Cdc42⁶ and Cdc42⁴/Cdc42⁶, demonstrated that both combinations produce over 70% embryonic lethality, even when outcrossed to wild-type males. Given the Mendelian expectation that 25% of the progeny will be hemizygous for the lethal allele, the high embryonic lethality indicates that Cdc42 is maternally contributed to the embryo and is essential (Genova, 2000).

To examine the role of Cdc42 in embryogenesis, the phenotypes of embryos with reduced maternal Cdc42 function were examined. Cuticle preparations and staining with anti-Coracle, which specifically stains ectodermally derived epithelial cells, revealed epidermal defects in these embryos, prior to the process of dorsal closure, that first appeared at the onset of germ-band retraction. In the embryos that failed to hatch, germ-band retraction failed to proceed to completion, leaving embryos open on the dorsal side. In addition, holes frequently appeared in the embryonic epidermis along the ventral midline. In extreme cases, possibly corresponding to embryos that also lacked zygotic Cdc42 function, the ventral epidermis was observed to tear apart along almost the entire anterior-posterior axis of the retracting embryo (Genova, 2000).

A previous study using a dominant activated Cdc42 allele has proposed that Cdc42 functions in neuronal cells to regulate neurite outgrowth (Luo, 1994). To determine if Cdc42 is required for normal neurite formation, embryos with reduced maternal Cdc42 contribution were examined for defects in the central and peripheral nervous systems. Because the embryonic defects observed were not completely penetrant and some embryos (<30%) completed embryogenesis normally, embryos with obvious germ-band retraction and epidermal defects were selected as an indication of reduced Cdc42 function. Patterns of neuronal differentiation, as determined by mAb 22C10 and anti-horseradish peroxidase (anti-HRP), were largely normal, even in areas where the integrity of the overlying epidermis was disrupted. In certain embryos defects in the central nervous system (CNS) such as incomplete formation and midline fusion of the longitudinal commissures were observed. However, in all embryos the axons of the peripheral nervous system as detected by antibody 22C10 appeared to extend properly. The defects observed in the CNS could represent a requirement for Cdc42 function in this tissue or they could be secondary to the other embryonic defects, such as failure in germband retraction and epidermal holes. In either case, the formation of axon commissures in the CNS and the apparently correct projection of neurites in the peripheral nervous system suggest that Cdc42 function is not essential for neurite outgrowth. Alternatively, it is possible that the requirement for Cdc42 function is higher in the epidermis than in other tissues or that tissues of the nervous system contain greater amounts of the maternal Cdc42 product (Genova, 2000).

The function of Cdc42 in postembryonic development was examined using analysis of phenotypes caused by partial loss-of-function mutations and somatic mosaic analysis of lethal mutations. Flies homozygous for weak Cdc42 alleles or carrying combinations of weak and strong alleles displayed distinct visible phenotypes. Scanning electron micrographs of Cdc42 mutant eyes revealed minor flaws in the ommatidial array, primarily in the dorsal posterior quadrant. In this region, occasional fusions of the ommatidia were observed and frequent loss or duplication of bristles. Although these phenotypes suggest possible defects in the structure of ommatidial units, examination of histological sections from Cdc42 mutant eyes revealed that all of the photoreceptor cells are present and in the proper orientation (Genova, 2000).

Cdc42 mutant adults also possessed extra crossveins on the wing between veins II and III and veins III and IV. Some allelic combinations displayed blisters in the wings where the dorsal and ventral surfaces were not fully apposed. Extra crossveins were also observed in 12% of flies heterozygous for Cdc42¹, consistent with the assertion that this allele has dominant negative properties. To examine the function of Cdc42 at the cellular level, somatic mosaic clones were generated to produce cells homozygous for Cdc42 lethal alleles in the wing imaginal discs. Clones generated early in larval development, at 36 h AEL, did not survive to the wandering third-instar stage, leaving only sister clones that mark where a recombination event occurred. Mutant clones that were generated later in larval development, at 72 h AEL, were smaller than their sister clones at the end of larval development. In these discs, mutant clones that occurred in the notum were only slightly reduced in size relative to their sister clones, whereas the mutant clones generated in the blade were either absent or much smaller than their sisters. This clonal pattern is characteristic of a cell competition effect and suggests that Cdc42 mutant cells are at a proliferation disadvantage relative to their wild-type neighbors (Genova, 2000).

As in the wing discs, Cdc42² clones generated in the eye at 36 h AEL were not observed in wandering third-instar larvae. Clones generated throughout the eye later in development, at 72 or 96 h AEL, did survive to the wandering third-instar stage, but did not survive to the adult stage. In histological sections of such eyes, mild pattern disruptions were observed around small scars near the sister clones, but no mutant cells had differentiated into photoreceptors or other recognizable cell types. To overcome the implied competition disadvantage of Cdc42 mutant cells and to examine their ability to differentiate adult structures, eye clones were generated in a Minute background. Control clones generated in the developing eye at 72 or 96 h AEL in a Minute background grew to encompass the majority of the eye. In contrast, clones of Cdc42 mutant cells failed to produce adult ommatidia and instead left scars on the eye. This result suggests that, in this case, the competition effect is due to a cause other than impaired proliferation (Genova, 2000).

To examine the development of these clones, it was asked if, in a wild type background, Cdc42 mutant cells could initiate neuronal differentiation. As determined by anti-HRP staining for developing neurons, mutant clones generated in eye discs 72 h AEL initiated differentiation as photoreceptor cells in the larval eye imaginal disc. These results indicate that although Cdc42 function is not required to initiate neuronal cell fate in the eye, it is necessary to complete photoreceptor differentiation (Genova, 2000).

Previous studies using ectopically expressed dominant alleles of yeast CDC42 and Drosophila Cdc42 in flies have suggested that Cdc42 may have important functions during Drosophila oogenesis (Murphy, 1996). To examine the effects of loss of Cdc42 function in the somatic and germ-line cells of the developing egg chamber, Cdc42² clones were generated via mitotic recombination. Follicle cell clones had three highly penetrant phenotypes: loss of columnar cell shape, formation of multiple layers of cells at the posterior end of the egg chamber, and apparent fusion of adjacent egg chambers. Germ-line clones displayed minor defects in actin filament assembly and, unexpectedly, defects in a subset of the follicle cells (Genova, 2000).

At stage 9 of egg chamber development, wild-type follicle cells change from a cuboidal cell shape to a more columnar shape. However, in mosaic egg chambers Cdc42 mutant cells remain cuboidal or irregular in cell shape while the wild-type cells abutting the mutant clone become columnar. Anti-Armadillo staining indicated that although the cells are abnormally shaped, the adherens junctions remain intact and cell polarity is maintained. Other apical markers, such as filamentous actin and Notch, also indicated that the apical- basal polarity is normal (Genova, 2000).

Clones of Cdc42 mutant follicle cells appear to lose the characteristics of a monolayered epithelium and form multiple layers of cells, primarily at the poles of the egg chamber. Mutations in alpha Spectrin, which produce a similar multilayered phenotype, have been shown to cause overproliferation in the follicle cells. To determine if the Cdc42 phenotype is due to overproliferation or alternatively is caused by redistribution of mutant cells to the posterior end of the egg chamber, follicle cell number was measured in egg chambers containing over 75% mutant follicle cells. These egg chambers did not appear to accumulate yolk in the oocyte and they degenerated by stage 9. The average follicle cell number in egg chambers containing Cdc42 mutant cells was not significantly different from that of wild-type egg chambers. In addition, cell division, as determined using antiphosphohistone H3, an antibody marker for mitotic cells, ceases at stage 6 in mutant clones as it does in wild-type cells. These results indicate that Cdc42 mutant follicle cells do not overproliferate, but rather display an inability to form a normal, single-layered epithelium (Genova, 2000).

A third phenotype observed in follicle cell clones that comprised the entire egg chamber was that egg chambers often appeared abnormally large, containing germ-line cells of two distinct size classes. These characteristics indicate that adjacent cysts may have fused or failed to separate during oogenesis. To examine this phenotype further, egg chambers were stained with anti-PS1 integrin, a stalk-cell-specific marker. Fused egg chambers appeared to lack stalk cells, suggesting that Cdc42 function is necessary for stalk cell differentiation or placement at the poles of egg chambers (Genova, 2000).

In the female germ line, highly organized bundles of cytoplasmic filaments are rapidly polymerized at stage 10b in the nurse cells. To examine Cdc42 localization during the polymerization of these actin filaments, a Myc-tagged Cdc42¹ transgene was constructed whose expression was regulated by the Drosophila Ubiquitin promoter. This transgene rescued lethal Cdc42 alleles, indicating that the addition of the Myc epitope tag does not affect Cdc42 function. In nurse cells prior to and during stage 10b, Myc-tagged Cdc42 was observed throughout the cytoplasm and was concentrated at the plasma membrane. In addition, it was observed that during stage 10b Cdc42 accumulates in regions where the cytoplasmic actin filaments intersect the plasma membrane. This localization pattern suggests that Cdc42 is recruited to sites on the plasma membrane where actin assembly is occurring (Genova, 2000).

Given the localization of Myc-tagged Cdc42 to the base of cytoplasmic actin filaments, it is reasonable to propose that Cdc42 function is required for the formation of these filaments. To test this hypothesis, ovaries were examined from mutant germ-line clones, produced using the ovo^D1 dominant, female-sterile mutation and in females carrying hypomorphic combinations of alleles. In egg chambers containing Cdc42 mutant germ-line clones, the characteristic cytoplasmic actin filaments still form and have essentially normal morphology. However, compared to wild-type nurse cells, Cdc42 mutant nurse cells appear to have fewer cytoplasmic actin filaments. In addition, those actin filaments directly associated with the ring canals appeared longer than normal. Dumping of nurse cell contents into the oocyte is delayed and does not appear to proceed to completion. Although these egg chambers fail to produce functional oocytes, staining with anti-Vasa antibody indicated that their overall anterior-posterior polarity is properly established (Genova, 2000).

Recent studies have shown that numerous interactions occur between the germ-line cells and surrounding somatic follicle cells during oogenesis. To determine if any of these interactions depend on Cdc42 function in the germ line, egg chambers containing mutant germ-line cells and wild-type follicle cells (as determined by the structure of the follicular epithelium) were examined. Staining with anti-PS1 showed a greater number of stalk cells adjacent to Cdc42 mutant germ-line clones than in wild-type controls. Interestingly, a study of the Drosophila toucan gene, which encodes a novel protein, has shown that this gene also functions in the germ line to regulate stalk cell number. This result indicates that interactions between the germ line and the soma are important for the differentiation of the stalk cells and that Cdc42 function is required in the germ line for this process (Genova, 2000).

Cdc42 was initially isolated as a cell division mutation that affects the process of budding in the yeast S. cerevisiae (Adams, 1990). It has also been implicated in the proper formation of the actin and myosin contractile ring during cytokinesis (Drechsel, 1997). Taken together, these results suggest that Cdc42 is essential for cell division in all organisms. However, the results presented in this study indicate that this hypothesis is incorrect: Cdc42 function is not required for proliferation of the follicular epithelium. In the ovary Cdc42 mutant follicle cell clones induced 6 days prior to dissection were frequently observed, that were large enough to comprise almost an entire ovariole. The size of the follicle cell clones and the extent of the ovariole that they cover indicate that the recombination was induced in stem cells that subsequently underwent many rounds of cell division. Given the length of time that these clones persisted and their size, it is unlikely that perdurance played a role in the survival of the mutant cells. Thus, Cdc42 does not appear to be required directly for cytokinesis or other aspects of cell proliferation (Genova, 2000).

Cdc42 mutant epithelial cells display phenotypes indicating an inability to undergo changes in cell shape that normally occur during embryogenesis, oogenesis, and imaginal development. At stage 9 during oogenesis, follicle cells undergo a transition from cuboidal to columnar cell shape. Simultaneously, most of the follicle cells migrate toward the posterior end of the egg chamber, leaving only a few specialized squamous cells covering the nurse cells at the anterior end of the egg chamber. In contrast to wild-type cells, Cdc42² cells fail to undergo this change of cell shape and instead remain more cuboidal or irregular in shape. Defects in cell elongation were also observed when dominant negative Cdc42 was expressed during Drosophila development (Eaton, 1995, 1996; Luo, 1994). Although improperly shaped, Cdc42 mutant follicle cells still migrate, which results in multiple layers of cuboidal cells at the posterior end of the egg chamber. This phenotype indicates that while still competent to migrate, mutant follicle cells are unable to undergo the changes in cell shape necessary for proper epithelial organization. Results using somatic mosaic analysis in the imaginal epithelium differ in some respects from those in the follicular epithelium. Although very large clones of mutant cells have been observed in the follicular epithelium, such clones were never found in the imaginal discs. Instead, Cdc42 mutant clones are lost rapidly from the imaginal epithelium in a manner resembling cell competition. The inability of cells to compete in the imaginal epithelium has been shown to be characteristic of cells that proliferate more slowly than their wild-type neighbors. However, the observation that Cdc42² cells in the follicular epithelium proliferate normally suggests that an inability to differentiate properly is the cause of clone loss. Consistent with this hypothesis, Cdc42 mutant clones induced in the background of a Minute mutation, which slows the growth of neighboring cells thereby rescuing clones with a proliferative disadvantage, still fail to survive to the adult stage. A previous study using ectopic expression of a dominant negative Cdc42 allele in wing imaginal disc cells has shown that these cells are unable to elongate properly to a columnar shape (Eaton, 1995), a phenotype similar to that which has been observed for Cdc42² follicle cells. Thus it is possible that cells that are unable to elongate properly to form the columnar shape that is typical of imaginal disc cells are eliminated from the epithelium by cell competition. If so, cell competition may represent a general response to eliminate cells that do not function normally, rather than a specific response to proliferation defects (Genova, 2000).

In the developing embryonic epithelium, phenotypes were also observed indicating a role for Cdc42 in epithelial morphogenesis. In embryos derived from females that are hypomorphic for Cdc42 function, early embryogenesis through germ-band extension occurs normally, presumably by using maternally encoded Cdc42. However, holes appear in the embryonic epithelium at the onset of germ-band retraction, particularly along the ventral midline. Epidermal cells along the ventral midline undergo extensive cell shape changes and rearrangements during germband retraction. Taken together, the phenotypes observed in developing Drosophila epithelial cells demonstrate a role for Cdc42 in cell elongation and other dynamic cell shape changes. In addition, the presence of epithelial holes may indicate that Cdc42 mutant cells are unable to either form or maintain normal adhesive contacts (Genova, 2000).

How does Cdc42 function in cell shape and morphogenesis? In epithelial cells, Cdc42 is essential for elongation into a columnar cell shape, a process that is likely to be dependent on actin filament assembly. Although the small size of Drosophila epithelial cells did not allow a direct analysis of cytoskeletal structure in Cdc42 mutant cells, the cortical actin filaments that form in the nurse cells during stage 10b of oogenesis are much larger and are readily observable. In Cdc42² nurse cells the number of cortical actin filaments is typically reduced relative to wild-type cells, but those that form appear morphologically normal. Myc-tagged Cdc42 protein localizes to the plasma membrane in these cells and is concentrated at the base of the clusters of cortical actin filaments. These results suggest that Cdc42 functions at the plasma membrane to facilitate the nucleation of actin filaments, but is not absolutely required for their formation (Genova, 2000).

This view of Cdc42 function in vivo agrees well with evidence from studies in vitro. Recent studies indicate that Cdc42 plays a primary role in the nucleation of new actin filaments by regulating the activity of an actin filament nucleating complex composed of the Wiskott-Aldrich syndrome protein (WASP: see Drosophila WASP) and the Arp2/3 complex (Rohatgi, 1999). Although Arp2/3 and WASP can nucleate new actin filaments in the absence of Cdc42, this activity increases dramatically in the presence of activated Cdc42 and phospholipids. Thus the reduction (rather than complete loss) of assembly of cortical actin filaments observed is consistent with the hypothesis that Cdc42 functions by regulating the rate of actin filament assembly, presumably in response to activation of an as yet unknown signaling pathway (Genova, 2000).

Genetic dissection of active forgetting in labile and consolidated memories in Drosophila

Different memory components are forgotten through distinct molecular mechanisms. In Drosophila, the activation of 2 Rho GTPases (Rac1 and Cdc42), respectively, underlies the forgetting of an early labile memory (anesthesia-sensitive memory, ASM) and a form of consolidated memory (anesthesia-resistant memory, ARM). This study dissected the molecular mechanisms that tie Rac1 and Cdc42 to the different types of memory forgetting. Two WASP family proteins, SCAR/WAVE and WASp, act downstream of Rac1 and Cdc42 separately to regulate ASM and ARM forgetting in mushroom body neurons. Arp2/3 complex, which organizes branched actin polymerization, is a canonical downstream effector of WASP family proteins. However, this study found that Arp2/3 complex is required in Cdc42/WASp-mediated ARM forgetting but not in Rac1/SCAR-mediated ASM forgetting. Instead, Rac1/SCAR may function with formin Diaphanous (Dia), a nucleator that facilitates linear actin polymerization, in ASM forgetting. The present study, complementing the previously identified Rac1/cofilin pathway that regulates actin depolymerization, suggests that Rho GTPases regulate forgetting by recruiting both actin polymerization and depolymerization pathways. Moreover, Rac1 and Cdc42 may regulate different types of memory forgetting by tapping into different actin polymerization mechanisms (Gao, 2019).

There are 3 major findings. First, 2 WASP family proteins, SCAR/WAVE and WASp, act as downstream effectors of Rac1-mediated ASM forgetting and Cdc42-mediated ARM forgetting, respectively. Second, although the Arp2/3 complex is a well-established effector that links activation of WASP family proteins to actin polymerization, it is only required in Cdc42/WASp-mediated ARM forgetting. Instead, formin Dia functions together with Rac1/SCAR in ASM forgetting. Third, feeding inhibitors of the Arp2/3 complex and Dia to fruit flies led to rather specific effects on ASM and ARM forgetting, raising the possibility of developing drugs on these molecular targets to treat memory-related diseases (Gao, 2019).

The effect of Rac1 on ASM forgetting has been tied to the activation of an actin depolymerization regulator cofilin presumably through a PAK/LIMK phosphorylation cascade. However, actin dynamics is a balanced play that requires continuous turnover between polymerization and depolymerization. It is not known whether signaling pathways regulating actin polymerization also play a role. There are 3 families of proteins that nucleate and promote actin polymerization, Arp2/3 complex, WH2-domain proteins, and formin. The finding that Arp2/3 complex and formin Dia function in ARM and ASM forgetting suggests that both actin polymerization and depolymerization pathways contribute to forgetting. How Arp2/3 complex and Dia separately contribute to ARM and ASM forgetting remains an open question. It is yet to be determined whether these proteins have different expression or subcellular locations in the MB neurons. However, it is interesting that Arp2/3 complex and formins are specialized in different types of actin polymerization (Gao, 2019).

In a working model, Cdc42 activates Arp2/3 complex via a canonical pathway (Cdc42/WASp/Arp2/3 complex), while Rac1-mediated ASM forgetting depends on SCAR/WAVE complex. This complex, in addition to SCAR/WAVE, includes at least 4 other members: Sra-1, Abi, HSPC300, and Kette. These additional members are thought to hold SCAR/WAVE in the complex in an inactive state, until GTP-bound Rac1 binds to Sra-1 and relieves the inhibition. On the other hand, the intact complex is essential for the stability of the SCAR/WAVE protein as well (i.e., failure to keep the intact complex can lead to SCAR degradation). This latter effect may explain the observation that RNAi knockdown of SCAR complex members has the same effect on inhibiting forgetting as the knockdown of SCAR. As a WASP family protein, SCAR/WAVE is able to associate with and activate Arp2/3 complex through its C-terminal region. However, RNAi knockdown of Arp2 and Arp3 and pharmacological inhibition of Arp2/3 complex specifically affects ARM forgetting, while no effects on ASM retention were observed. It is therefore proposed that Rac1/SCAR may function through Arp2/3 complex-independent mechanisms. SCAR/WAVE complex is reported to physically associates with Dia through one of its members, Abi, to regulate actin dynamics. Behavioral characterization of Dia knockdown and overexpression, as well as the genetic epistasis experiment, support the idea that Dia could be downstream of Rac1/SCAR in ASM forgetting. Details about the functional coordination between SCAR/WAVE and Dia therefore await further clarification (Gao, 2019).

RhoGAP19D inhibits Cdc42 laterally to control epithelial cell shape and prevent invasion

Cdc42-GTP is required for apical domain formation in epithelial cells, where it recruits and activates the Par-6-aPKC polarity complex, but how the activity of Cdc42 itself is restricted apically is unclear. This study used sequence analysis and 3D structural modeling to determine which Drosophila GTPase-activating proteins (GAPs) are likely to interact with Cdc42 and identified RhoGAP19D as the only high-probability Cdc42GAP required for polarity in the follicular epithelium. RhoGAP19D is recruited by α-catenin to lateral E-cadherin adhesion complexes, resulting in exclusion of active Cdc42 from the lateral domain. rhogap19d mutants therefore lead to lateral Cdc42 activity, which expands the apical domain through increased Par-6/aPKC activity and stimulates lateral contractility through the myosin light chain kinase, Genghis khan (MRCK). This causes buckling of the epithelium and invasion into the adjacent tissue, a phenotype resembling that of precancerous breast lesions. Thus, RhoGAP19D couples lateral cadherin adhesion to the apical localization of active Cdc42, thereby suppressing epithelial invasion (Fic, 2021).

The form and function of epithelial cells depends on their polarization into distinct apical, lateral, and basal domains by conserved polarity factors. This polarity is then maintained by mutual antagonism between apical polarity factors such as atypical PKC (aPKC) and lateral factors such as Lethal (2) giant larvae (Lgl) and Par-1. While many aspects of the polarity machinery are now well understood, it is still unclear how the apical domain is initiated and what role cell division control protein 42 (Cdc42) plays in this process. Cdc42 was identified for its role in establishing polarity in budding yeast, where it targets cell growth to the bud tip by polarizing the actin cytoskeleton and exocytosis toward a single site. It has subsequently been found to function in the establishment of cell polarity in multiple contexts. For example, Cdc42 recruits and activates the anterior PAR complex to polarize the anterior-posterior axis in the Caenorhabditis elegans zygote and the apical-basal axis during the asymmetric divisions of Drosophila neural stem cells. Cdc42 also plays an essential role in the apical-basal polarization of epithelial cells, where it is required for apical domain formation. Cdc42 is active when bound to GTP, which changes its conformation to allow it to bind downstream effector proteins that control the cytoskeleton and membrane trafficking. An important Cdc42 effector in epithelial cells is the Par-6-aPKC complex. Par-6 binds directly to the switch 1 region of Cdc42 GTP through its semi-CRIB domain (Cdc42 and Rac interactive binding). This induces a change in the conformation of Par-6 that allows it to bind to the C-terminus of another key apical polarity factor, the transmembrane protein Crumbs, which triggers the activation of aPKC's kinase activity. As a result, active aPKC is anchored to the apical membrane, where it phosphorylates and excludes lateral factors, such as Lgl, Par-1, and Bazooka (Baz). In addition to this direct role in apical-basal polarity, Cdc42 also regulates the organization and activity of the apical cytoskeleton through effectors such as neuronal Wiskott-Aldrich syndrome protein (N-WASP), which promotes actin polymerization, and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK; Genghis khan [Gek] in Drosophila), which phosphorylates the myosin regulatory light chain to activate contractility (Fic, 2021).

This crucial role of active Cdc42 in specifying the apical domain raises the question of how Cdc42-GTP itself is localized apically. In principle, this could involve activation by Cdc42 guanine nucleotide exchange factors (Cdc42GEFs) that are themselves apical or lateral inactivation by Cdc42GAPs. The Cdc42GEFs Tuba, intersectin 2, and Dbl3 have been implicated in activating Cdc42 in mammalian epithelia. Only Dbl3 localizes apical to tight junctions, however, as Tuba is cytoplasmic and enriched at tricellular junctions and intersectin 2 localizes to centrosomes. Thus, GEF activity may not be exclusively apical, suggesting that it is more important to inhibit Cdc42 laterally. Although nothing is known about the role of GAPs in restricting Cdc42 activity to the apical domain of epithelial cells, this mechanism plays an instructive role in establishing radial polarity in the blastomeres of the early C. elegans embryo. In this system, the Cdc42GAP PAC-1 is recruited by the cadherin adhesion complex to sites of cell-cell contact, thereby restricting active Cdc42 and its effector the Par-6-aPKC complex to the contact-free surface (Fic, 2021).

This study has analyzed the roles of Cdc42GAPs in epithelial polarity using the follicle cells that surround developing Drosophila egg chambers as a model system. By generating mutants in a number of candidate Cdc42GAPs, this study identified the Pac-1 orthologue, RhoGAP19D, as the GAP that restricts active Cdc42 to the apical domain. In the absence of RhoGAP19D, lateral Cdc42 activity leads to an expansion of the apical domain and a high frequency of epithelial invasion into the germline tissue, a phenotype that mimics the early steps of carcinoma formation (Fic, 2021).

In the absence of RhoGAP19D, both N-WASP and Gek are recruited to the lateral membrane, indicating that Cdc42 is ectopically activated there. This implies that RhoGAP19D is the major Cdc42GAP that represses Cdc42 laterally, because no other GAPs can compensate for its loss. This also suggests that the GEFs that activate Cdc42 are not restricted to the apical domain and can turn it on laterally once this repression is removed. This is consistent with the identification of multiple vertebrate GEFs with different localizations that contribute to apical Cdc42 activation. The current results therefore identify RhoGAP19D as a new lateral polarity factor. This leads to a revised network of polarity protein interactions in which RhoGAP19D functions as the third lateral factor that antagonizes the activity of apical factors, alongside Lgl, which inhibits aPKC, and Par-1, which excludes Baz/Par-3 (Fic, 2021).

The function of RhoGAP19D is very similar to that of its orthologue PAC-1, which inhibits Cdc42 at sites of cell contact in early C. elegans blastomeres to generate distinct apical and basolateral domains. Both RhoGAP19D and PAC-1 are recruited to the lateral domain by E-cadherin complexes, although the exact mechanism is slightly different. RhoGAP19D recruitment is strictly dependent on α-catenin, which links it through β-catenin to the E-cadherin cytoplasmic tail, whereas α-catenin (HMP-1) and p120-catenin (JAC-1) play partially redundant roles in recruiting PAC-1 to E-cadherin (HMR-1) in the worm. Nevertheless, in both cases, the recruitment of the Cdc42GAP translates the spatial cue provided by the localization of cadherin to sites of cell-cell contact into a polarity signal that distinguishes the lateral from the apical domain. Classic work on the establishment of polarity MDCK cells grown in suspension has revealed that the recruitment of cadherin (uvomorulin) to sites of cell-cell contact is the primary cue that drives the segregation of apical proteins from basolateral proteins. Furthermore, the expression of E-cadherin in unpolarized mesenchymal cells is sufficient to induce this segregation, although the mechanisms behind this process are only partially understood. The observation that RhoGAP19D directly links cadherin adhesion to the polarity system in epithelial cells extends the results of Klompstra (2015) in early blastomeres, strongly suggesting that PAC-1/RhoGAP19D plays an important role in the first steps in epithelial polarization (Fic, 2021).

Although PAC-1 and RhoGAP19D perform equivalent functions in early blastomeres and epithelial cells, there is one important difference between their mutant phenotypes. In pac-1 mutants, Par-6 and aPKC are mislocalized to the contacting surfaces of C. elegans blastomeres where Cdc42 is ectopically active. By contrast, Par-6 and aPKC are not mislocalized laterally in rhogap19d mutant Drosophila epithelial cells, even though lateral Cdc42-GTP does recruit two other Cdc42 effectors, N-WASP and Gek. Thus, lateral Cdc42 activity is sufficient to recruit Par-6/aPKC to the lateral domain in early blastomeres, but not in epithelial cells. Instead, it was observed that lateral Cdc42 activity in rhogap19d mutant follicle cells acts at a distance to expand the size of the apical domain. A likely explanation for this difference is the presence of Crumbs in epithelial cells. The interaction between Cdc42-GTP and Par-6 alters the conformation of Par-6 so that it can bind to Crumbs, which anchors the Par-6-aPKC complex to the apical membrane and activates aPKC's kinase activity. Although Par-6 presumably binds to Cdc42 laterally in rhogap19D mutants and undergoes the conformational change, it cannot be anchored laterally in the absence of Crumbs. This activated Par-6-aPKC complex can then diffuse until it is captured by Crumbs in the apical domain, thereby increasing apical aPKC activity, providing an explanation for why the apical domain expands in rhogap19d mutant cells. C. elegans has three Crumbs orthologues, but removal of all three simultaneously has no effect on viability or polarity. Thus, in contrast to Drosophila epithelial cells, C. elegans Crumbs proteins are not required for Par-6/aPKC localization and activation, suggesting that some other mechanism, such as Cdc42 binding, is sufficient to activate aPKC (Fic, 2021).

If the failure of active Cdc42 to recruit aPKC laterally in rhogap19d mutant cells is due to the absence of Crumbs in this region, there must be a mechanism to exclude Crumbs from the lateral domain. One proposed mechanism depends on Yurt (Moe and EPB41L5 in vertebrates), which is restricted to the lateral domain by aPKC and binds to Crumbs to antagonize its activity. However, no lateral recruitment of aPKC was observed in rhogap19d;yurt double-mutant cells. Thus, there must be some parallel mechanism that excludes Crumbs, Par-6, and/or aPKC from the lateral domain (Fic, 2021).

Although loss of RhoGAP19D only leads to a partial disruption of polarity, it causes the follicular epithelium to invade the adjacent germline tissue with 40% penetrance. This invasive behavior is not driven by an epithelial-to-mesenchymal transition, because the cells retain their apical adherens junctions and epithelial organization. Instead, the deformation of the epithelium seems to be driven by the combination of an increase in lateral contractility and an expansion of the apical domain, because reducing the dosage of Gek, which activates myosin II to drive the contractility, significantly reduces the frequency of this phenotype, as does halving the dosage of any of the apical polarity factors. The expansion of the apical domain makes the domain too long for the cells to adopt the lowest-energy conformation, giving them a tendency to become wedge shaped, which could drive the evagination. It is also possible that buckling of the epithelium contributes to invasion. Recent work has shown that epithelial monolayers under compressive stress and constrained by a rigid external scaffold have a tendency to buckle inward. The follicular cell layer is surrounded by an ECM that constrains the shape of the egg chamber and that should therefore resist expansion. In addition, the pulses of lateral contractility are likely to generate compressive stress because transiently reducing cell height while maintaining a constant volume will increase the cells' cross-sectional area, thereby exerting a pushing force on the neighboring cells. This compression coupled to the tendency to become wedge shaped due to apical expansion could therefore trigger the rare buckling events that initiate invasion. In support of this view, lateral contractility has been shown to drive the folding of the imaginal wing disc between the prospective hinge region and the pouch. This phenotype provides an example of how a partial disruption of polarity can induce cell shape changes that lead to major alterations in tissue morphogenesis (Fic, 2021).

The rhogap19d phenotype resembles the defects earliest observed in the development of ductal carcinoma in situ. In flat epithelial atypia (FEA), the ductal cells are still organized into an epithelial layer, but they display apical protrusions that are strongly labeled by the apical polarity factor Par-6. This suggests that the apical domain has expanded and bulges out of the cell, just as was observed in the rhogap19d mutant follicular cells. In the next stage, atypical ductal hyperplasia (ADH), the ductal cells start to invade the lumen of the duct while retaining aspects of normal apical-basal polarity. This again resembles the invasive phenotype of rhogap19d mutants, although overproliferation of the ductal cells probably also contributes to invasion in this case. Thus, these abnormalities, which can sometimes progress to ductal carcinoma in situ and breast cancer, mirror the effects of lateral Cdc42 activation. The RhoGAP19D human orthologues, ARHGAP21 and ARHGAP23, have been shown to bind directly to α-catenin and localize to cell-cell junctions. Furthermore, low expression of ARHGAP21 or ARHGAP23 correlates with reduced survival rates in several cancers of epithelial origin. It would therefore be interesting to determine whether these orthologues perform the same functions in epithelial polarity as RhoGAP19D and if their loss contributes to tumor development (Fic, 2021).

Filopodia-based contact stimulation of cell migration drives tissue morphogenesis

Cells migrate collectively to form tissues and organs during morphogenesis. Contact inhibition of locomotion (CIL) drives collective migration by inhibiting lamellipodial protrusions at cell-cell contacts and promoting polarization at the leading edge. This study reports a CIL-related collective cell behavior of myotubes that lack lamellipodial protrusions, but instead use filopodia to move as a cohesive cluster in a formin-dependent manner. Genetic, pharmacological and mechanical perturbation analyses were performed to reveal the essential roles of Rac2, Cdc42 and Rho1 in myotube migration. These factors differentially control protrusion dynamics and cell-matrix adhesion formation. Active Rho1 GTPase localizes at retracting free edge filopodia and Rok-dependent actomyosin contractility does not mediate a contraction of protrusions at cell-cell contacts, but likely plays an important role in the constriction of supracellular actin cables. Based on these findings, it is proposed that contact-dependent asymmetry of cell-matrix adhesion drives directional movement, whereas contractile actin cables contribute to the integrity of the migrating cell cluster (Bischoff, 2021).

The ability of cells to migrate as a collective is crucial during tissue morphogenesis and remodeling. The molecular principles of collective cell migration share features with the directed migration of individual cells. The major driving forces in migrating single cells are Rac-mediated protrusions of lamellipodia at the leading edge, formed by Arp2/3 complex-dependent actin filament branching and Rho-dependent actomyosin-driven contraction at the cell rear. Cells can migrate directionally in response to a variety of chemical cues, recognized by cell surface receptors that initiate downstream signaling cascades controlling the activity or recruitment of Rho GTPases. Directional cell locomotion is also controlled by mechanical stimuli such as upon cell-cell contact. A well-known phenomenon is contact inhibition of locomotion (CIL), whereby two colliding cells change direction after coming into contact. Mechanistic evidence has been obtained of how CIL might act in vivo as the driving force to polarize neural crest cells that derived from the margin of the neural tube and disperse by migration during embryogenesis (Bischoff, 2021).

In neural crest cells, CIL involves distinct stages of cell behavior including cell-cell contact, protrusion inhibition, repolarization, contraction, and migration away from the collision. The initial cell-cell contact requires the formation of transient cadherin-mediated cell junctions. Once the cells come in close contact, a disassembly of cell-matrix adhesion near the cell-cell contact and the generation of new cell-matrix adhesions at the free edge occur. Such mechanical crosstalk between N-cadherin-mediated cell-cell adhesions and integrin-dependent cell-matrix adhesions has been recently described in vivo during neural crest cell migration in both Xenopus and zebrafish embryos. However, the loss of cell-matrix adhesions at cell contacts alone is not sufficient to drive CIL. A subsequent repolarization of the cells away from the cell-cell contact and thereby the generation of new cell-matrix adhesions and protrusions at the free edge are required to induce cell migration away from the collision. In neural crest cells, this depends on the polarized activity of the two Rho GTPases, Rac1 and RhoA. A model of CIL has been proposed in which a contact-dependent intracellular Rac1/RhoA gradient is formed that generates an asymmetric force driving directed cell migration. N-cadherin binding triggers a local increase of RhoA and inhibits Rac1 activity at the site of contact. Thus, Rac1-dependent protrusions become biased to the opposite end of the cell-cell contact and cells migrating away from the collision (Bischoff, 2021).

Overall, CIL has been successfully used to explain contact-dependent collective migration of loose clusters of mesenchymal cells such as neural crest cells and hemocytes, but it is still unclear whether mechanisms governing CIL might also contribute to the migratory behavior of cohesive cell clusters or epithelia (Bischoff, 2021).

Using an integrated live-cell imaging and genetic approach, this study identified a CIL-related, contact-dependent migratory behavior of highly cohesive nascent myotubes of the Drosophila testis. Myotubes lack lamellipodial cell protrusions, but instead form numerous large filopodia generated at both N-cadherin-enriched cellular junctions at cell-cell contacts and integrin-dependent cell-matrix sites at their free edge. Filopodia-based myotube migration requires formins and the Rho family small GTPases Rac2, Cdc42, and RhoA, whereas the Arp2/3 complex and its activator, the WAVE regulatory complex (WRC), seem only to contribute to filopodia branching. Rac2 and Cdc42 differentially control not only protrusion dynamics but also cell-matrix adhesion formation. Unlike CIL, RhoA is not activated at cell-cell contacts, but rather gets locally activated along retracting protrusions. Genetic and pharmacological perturbation analysis further revealed an important requirement of Rho/Rok-driven actomyosin contractility in myotube migration (Bischoff, 2021).

In summary, a model is proposed in which N-cadherin-mediated contact-dependent asymmetry of cell-matrix adhesion acts as a major switch to drive cell movement toward the free space, whereas contractile actin cables contribute to the integrity of the migrating cell cluster (Bischoff, 2021).

The data imply that a contact-dependent migration mechanism acts as a driving force to polarize Drosophila myotubes and to promote their directional movement along the testes. A contact-stimulated migration has been already observed in cultured cells many years ago, but the molecular mechanisms underlying this phenomenon has been never analyzed in more detail. It has been observed that both primary neural crest cells and two neural crest-derived cell lines barely moved when isolated in suspension, but could be stimulated up to 200-fold to migrate following contact with migrating cells. This process might help to ensure the cohesion and coordination of collectively migrating myotubes to form dense muscular sheets in the walls of developing hollow organs. Those muscle fibers that race ahead will immediately cease migration when they lose contact with their neighbors. That is exactly what was observed in the current experiments. After ablation, an isolated myotube awaits restimulation by the other cells of the migrating cluster. Consistently, reduced N-cadherin function promotes single-cell migration toward the free space at the expense of collective directionality. The contact-dependent behavior of myotubes also resembles CIL, a well-characterized phenomenon. CIL regulates the in vivo collective cell migration of mesenchymal cells such as neural crest cells by inhibiting protrusions forming within the cluster at cell-cell edges and by driving actin polymerization at their free edge (Bischoff, 2021).

Different from neural crest cells, myotubes did not migrate as loose cohorts, but maintain cohesiveness (see Comparison between filopodia-based and lamellipodia-based cell migration). In the context of more-adhesive cells, a CIL-related mechanism, termed frustrated CIL has been proposed by which cell-cell junctions can determine the molecular polarity of a collectively migrating epithelial sheet. Evidence has been provided that cell-cell junctions determine the molecular polarity through a network of downstream effectors that independently control Rac activity at the cell-free end and Rho-dependent myosin II light chain activation at cell-cell junctions (Ladoux, 2017; Desai, 2009). At the first glance, myotubes do not show an obvious polarized cell morphology with prominent polarized protrusions. Instead, myotubes form numerous competing protrusions in all directions. However, protrusions pointing to the free space preferentially form more stable cell-matrix adhesions as anchorage sites for forward protrusions, whereas the lifetime of cell-matrix adhesions at cell-cell contacts is decreased. Thus, a contact-dependent asymmetry in matrix adhesion dynamics seems to be important for the directionality of migrating myotubes, a molecular polarity that has been also found in neural crest cells undergoing CIL. Only when one of the adhesions of competing protrusions disassembles, pulling of the cell body toward the competing protrusions might contribute to symmetry breaking and directionality of collective migration (Bischoff, 2021).

Evidence is provided for a differential requirement of the Rho GTPases, Rac2, and Cdc42 in regulating cell-matrix adhesion. cdc42 knockdown cells formed less cohesive clusters and showed a significant increase of cell-matrix adhesion lifetime probably due to a decrease cell-matrix adhesion turnover. In contrast, Rac2 depletion resulted in a prominent loss of cell-matrix adhesions, a phenotype that has already been described in Rac1-/- mouse embryonic fibroblasts. Thus, a model is proposed in which cell-matrix adhesions are downregulated at N-cadherin-dependent cell-cell contacts, a process that requires Cdc42 functions. To finally test whether a contact-dependent reduction of cell-matrix adhesion in filopodia is sufficient to explain the observed collective cell behavior, a simplified simulation model was developed with a few rules governing cell behavior such as protrusive filopodia, matrix adhesion, cell-cell adhesion, and membrane resistance. Unlike comparable computer models, single cells do not possess directional information. A cell's position is defined by the geometric center of all its filopodia, whose emergence/disappearance/elongation causes translation of the centroid, perceived as motion. Upon cell-cell contact, filopodia lose their cell-matrix adhesion and thereby their grip on the ECM, but keep connections through cell-cell adhesions. These adhesions are recognized by both contributing cells to calculate their respective centroids. Using these simple rules, it was possible to model myotube collective migration, provided that cells are positioned in a confined area mimicking the unfolded testis surface. If filopodia disappear directly after contact, cells exhibit a different cell behavior that is very reminiscent of CIL. This simplified model further confirms the observation that local regulation of cell-matrix adhesion suffices to drive collective motility (Bischoff, 2021).

Actomyosin function ensures the integrity of cohesive myotube cluster during migration Myotube migration also requires Rho1 the Drosophila homolog of RhoA. Different from cells undergoing CIL, in migrating myotubes activated Rho1 was not enriched at cell-cell contacts between myotubes, but rather localized as local pulses along retracting filopodial protrusions at free edges. The effects of tensile forces have to be addressed separately in the future, by establishing one of the many existing force measurement techniques such as transition force microscopy or using in vivo FRET-based tensions sensors in this system. Loss of Rok activity, sqh, and zip phenocopies rho1 knock down suggesting that a canonical pathway controls myotube migration in which Rho1 acts through Rok kinase to activate myosin II contractility. This finding supports the notion that in testis myotubes, unlike many other cell types, locally restricted Rho-GTPase regulation outweighs global Rac/Rho regulation along the cell-rear axis to achieve directionality. Previous studies demonstrated that myosin II-dependent contraction is essential for coordinating the CIL response in colliding cells. In myotube migration, Rok-dependent actomyosin contraction seems to be not required to drive the myotube cluster forward, but rather contractile actin cables contribute to the integrity of the migrating cell cluster. Thus, myotube cluster behave more like a collectively migrating monolayered epithelial sheet during gap closure. While myotubes migrate into any given free space, they leave larger gaps within the cell sheet surrounded by prominent circumferential actin cables. Constriction of these supracellular actin cables necessarily might lead to gap closure observed in wild type, but not in cells defective for RhoRok-driven actomyosin contractility (Bischoff, 2021).

Efficient mesenchymal cell migration on two-dimensional surfaces is thought to require the Arp2/3 complex generating lamellipodial branched actin filament networks that serve a major engine to push the leading edge forward (Bischoff, 2021).

Interestingly, epithelial and mesenchymal cells form more filopodia when the Arp2/3 complex is absent. Under these conditions, mesenchymal cells lack lamellipodia and adopt a different mode of migration only using matrix-anchored filopodial protrusions. The data further provide evidence for a filopodia-based cell migration in a physiological context during morphogenesis. This migration mode largely depends on formin as central known actin nucleators generating filopodia. The data also suggest that the Arp2/3 and its activator, the WRC, contribute to a more efficient myotube migration by promoting filopodia branching, and thereby increasing the number of cell-matrix adhesions, thus increased anchorage sites. Overall, filopodia-based migration enables the cell to regulate discrete subunits of membrane protrusions as an answer to the environment. The sum of filopodial protrusions adds up to a net cell locomotion that occurs similarly during lamellipodial migration. Filopodial matrix adhesion complexes not only provide anchorage sites, but also allow cells to directly restructure their microenvironment by membrane-bound matrix proteases. There is indeed increasing clinical evidence suggesting filopodia play a central role in tumor invasion. Similar to invading cancer cells myotubes rather migrate through a 3D microenvironment composed of extracellular matrix restricted by pigment cells from the outside of the testis. Thus, it will be interesting to determine to what extent extracellular matrix restructuring by metalloproteinases is required for myotube migration (Bischoff, 2021).

Taken together, the data suggest that contact-stimulated filopodia-based collective migration of myotubes depends on a CIL-related phenomenon combining features and molecular mechanisms described in mesenchymal and epithelial sheet migration as well. A model is proposed in which contact-dependent asymmetry of cell-matrix adhesion acts as a major switch to drive directional motion toward the free space, whereas contractile actin cables contribute to the integrity of the migrating cell cluster (Bischoff, 2021).

GENE STRUCTURE

cDNA clone length - 1443

Bases in 5' UTR - 191

Exons - 3

Bases in 3' UTR - 676

PROTEIN STRUCTURE

Amino Acids - 191

Structural Domains

See SMART (Simple Modular Architecture Research Tool) for information on Rho-family small GTPases.

date revised: 23 June 2023

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