RhoA is required for monocyte tail retraction during transendothelial migration.

Transendothelial migration of monocytes is the process by which monocytes leave the circulatory system and extravasate through the endothelial lining of the blood vessel wall and enter the underlying tissue. Transmigration requires coordination of alterations in cell shape and adhesive properties that are mediated by cytoskeletal dynamics. We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration. By loading monocytes with C3, an inhibitor of RhoA, we found that RhoA was required for transendothelial migration. We then examined individual steps of transmigration to explore the requirement for RhoA in extravasation. Our studies showed that RhoA was not required for monocyte attachment to the endothelium nor subsequent spreading of the monocyte on the endothelial surface. Time-lapse video microscopy analysis revealed that C3-loaded monocytes also had significant forward crawling movement on the endothelial monolayer and were able to invade between neighboring endothelial cells. However, RhoA was required to retract the tail of the migrating monocyte and complete diapedesis. We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction. Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of beta2 integrin in the unretracted tails.     

Mechanotransduction of shear stress occurs through changes in VE-cadherin and PECAM-1 tension: Implications for cell migration

Recent work has shown that cadherins at cell-cell junctions bear tensile forces. Using novel FRET-based tension sensors, we showed first that in response to shear stress, endothelial cells rapidly reduce mechanical tension on vascular endothelial (VE)-cadherin. Second, we observed a simultaneous increase in tension on platelet endothelial cell adhesion molecule (PECAM)-1, induced by an interaction with vimentin. In this commentary, we discuss how our results fit with existing data on cadherins as important mediators of mechanotransduction, in particular, in cell migration where mechanical tension across cadherins may communicate the direction of movement. The ability of PECAM-1 to bear mechanical tension may also be important in other PECAM-1 functions, such as leukocyte transmigration through the endothelium. Additionally, our observation that vimentin expression was required for PECAM-1 tension and mechanotransduction of fluid flow suggests that intermediate filaments are capable of transmitting tension. Overall, our results argue against models where an external force is passively transferred across the cytoskeleton, and instead suggest that cells actively respond to extracellular forces by modulating tension across junctional proteins.     

An octapeptide in the juxtamembrane domain of VE-cadherin is important for p120ctn binding and cell proliferation

The cadherins are a family of adhesive proteins involved in cell-cell homophilic interactions. VE-cadherin, expressed in endothelial cells, is involved in morphogenesis, regulation of permeability, and cellular proliferation. The cytoplasmic tails of cadherins contain two major domains, the juxtamembrane domain that plays a role in the intercellular localization of the protein and also serves for binding of p120ctn, and a C-terminal domain that associates with beta- or gamma-catenin. A highly conserved region present in the juxtamembrane domain of the cadherins has been shown to be necessary for p120ctn binding in E-cadherin. Using a mutant VE-cadherin lacking a highly conserved octapeptide, we demonstrated that it is required for p120ctn binding to VE-cadherin as determined by immunoprecipitation and colocalization studies. By immunofluorescence, this mutant protein has a topographical distribution similar to that of the wild-type VE-cadherin and, therefore, we conclude that the topographical distribution of VE-cadherin is independent of this motif. In addition, although cell-cell association is present in cells expressing this mutant form of VE-cadherin, we found that the strength of adhesion is decreased. Finally, our results for the first time demonstrate that the interaction of VE-cadherin with p120 catenin plays an important role in cellular growth, suggesting that the binding of p120 catenin to cadherins may regulate cell proliferation.     

VE-cadherin is a critical molecule for trophoblast-endothelial cell interaction in decidual spiral arteries

Fetal cytotrophoblasts colonize the decidual spiral arteries during pregnancy and partially replace the endothelium by an as yet unknown mechanism. To clarify this issue, we cocultured trophoblast cells (TCs) and decidual endothelial cells (DECs) isolated from first trimester placentae and found by electron microscopic analysis that TCs adhered to DECs and migrated through the interendothelial junctions within 24 h. Since extravillous TCs were shown by FACS analysis to express vascular-endothelial (VE)-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM)-1, we investigated the role of these junctional molecules in TC adhesion to DECs and transendothelial migration of cytotrophoblasts. Both VE-cadherin and PECAM-1 were present at the contact sites between TCs and DECs in decidual sections. TC adhesion and migration were markedly inhibited by mAbs to VE-cadherin and marginally by mAb to PECAM-1. Increased expression of VE-cadherin was observed at the contact areas between TCs and DECs, whereas PECAM-1 was found to be redistributed from intercellular junctions. The induction of apoptosis of DECs by TCs, as the mechanism responsible for their replacement, was ruled out by the negative staining with TUNEL of DECs cocultured with TCs and the absence of DNA fragmentation. In conclusion, VE-cadherin is involved in transendothelial migration of TCs, and replacement of DECs by TCs is not the result of apoptosis.     

Junctional adhesion molecules are required for melanoma cell lines transendothelial migration in vitro

One of the main steps of metastasis is extravasation, a phenomenon well described in lymphocytes but remaining to be fully uncovered for melanoma. Junctional adhesion molecules (JAMs) control the transendothelial migration of leukocytes. To date, the role of the JAM proteins, notably JAM-A and JAM-C, has not been examined in melanoma. Here, we compared two melanoma tumor cell lines, A375 and SLM8 cells, the A375 cell line being four times more efficient than the SLM8 cells in the crossing of the endothelial monolayer. We show evidence of the differential expression of JAM-A and JAM-C in these cell lines with JAM-C mainly expressed in the A375 cell line, and JAM-A detected preferentially in the SLM8 cells. To further dissect the respective roles of these proteins, we used both siRNA and blocking antibodies to decrease JAM-A and JAM-C expression.     

Distribution of adherens junction mediated by VE-cadherin complex in rat spleen sinus endothelial cells

The splenic sinus endothelium regulates the passage of blood cells through the splenic cord. The goal of the present study was to assess the localization of vascular endothelial (VE)-cadherin, β-catenin, and p120-catenin in the sinus endothelial cells of rat spleen and to characterize the presence and distribution of adherens junction formation mediated by the cadherin-catenin complex. Immunofluorescent microscopy of tissue cryosections demonstrated that VE-cadherin, β-catenin, and p120-catenin were localized in the junctional regions of adjacent endothelial cells. Double-staining immunofluorescent microscopy for VE-cadherin and β-catenin revealed colocalization at junctional regions. Transmission electron microscopy of thin sections of sinus endothelial cells treated with Triton X-100 clearly showed adherens junctions within the plasma membrane. Adherens junctions were located at various levels in the lateral membranes of adjacent endothelial cells regardless of the presence or absence of underlying ring fibers. Immunogold electron microscopy revealed VE-cadherin, β-catenin, and p120-catenin in the juxtaposed junctional membranes of adjacent sinus endothelial cells. Double-staining immunogold microscopy for VE-cadherin and β-catenin and for VE-cadherin and p120-catenin demonstrated colocalization to the junctional membranes of adjacent endothelial cells. Immunolabeling was evident at various levels in the lateral junctional membranes and was intermittently observed in the sinus endothelium. These data suggest that adherens junctions, whose formation appears to be mediated by VE-cadherin-catenin complexes, probably regulate the passage of blood cells through the spleen. This work was supported by a Grant-in-Aid for Scientific Research (C), Japan     

ClC-2 is required for rapid restoration of epithelial tight junctions in ischemic-injured murine jejunum

Background and aims

Involvement of the epithelial chloride channel ClC-2 has been implicated in barrier recovery following ischemic injury, possibly via a mechanism involving ClC-2 localization to the tight junction. The present study investigated mechanisms of intestinal barrier repair following ischemic injury in ClC-2^−/− mice.

Methods

Wild type, ClC-2 heterozygous and ClC-2^−/− murine jejunal mucosa was subjected to complete ischemia, after which recovery of barrier function was monitored by measuring in vivo blood-to-lumen clearance of ³H-mannitol. Tissues were examined by light and electron microscopy. The role of ClC-2 in re-assembly of the tight junction during barrier recovery was studied by immunoblotting, immunolocalization and immunoprecipitation.

Results

Following ischemic injury, ClC-2^−/− mice had impaired barrier recovery compared to wild type mice, defined by increases in epithelial paracellular permeability independent of epithelial restitution. The recovering ClC-2^−/− mucosa also had evidence of ultrastructural paracellular defects. The tight junction proteins occludin and claudin-1 shifted significantly to the detergent soluble membrane fraction during post-ischemic recovery in ClC-2^−/− mice whereas wild type mice had a greater proportion of junctional proteins in the detergent insoluble fraction. Occludin was co-immunoprecipitated with ClC-2 in uninjured wild type mucosa, and the association between occludin and ClC-2 was re-established during ischemic recovery. Based on immunofluorescence studies, re-localization of occludin from diffuse sub-apical areas to apical tight junctions was impaired in ClC-2^−/− mice.

Conclusions

These data demonstrate a pivotal role of ClC-2 in recovery of the intestinal epithelium barrier by anchoring assembly of tight junctions following ischemic injury.     

Functional significance of octameric RuvA for a branch migration complex from Thermus thermophilus

The RuvAB complex promotes migration of Holliday junction at the late stage of homologous recombination. The RuvA tetramer specifically recognizes Holliday junction to form two types of complexes. A single tetramer is bound to the open configuration of the junction DNA in complex I, while the octameric RuvA core structure sandwiches the same junction in complex II. The hexameric RuvB rings, symmetrically bound to both sides of RuvA on Holliday junction, pump out DNA duplexes, depending upon ATP hydrolysis. We investigated functional differences between the wild-type RuvA from Thermus thermophilus and mutants impaired the ability of complex II formation. These mutant RuvA, exclusively forming complex I, reduced activities of branch migration and ATP hydrolysis, suggesting that the octameric RuvA is essential for efficient branch migration. Together with our recent electron microscopic analysis, this finding provides important insights into functional roles of complex II in the coordinated branch migration mechanism.     

Endothelial intracellular Ca2+ release following monocyte adhesion is required for the transendothelial migration of monocytes

Although molecular changes accompanying leukocyte extravasation have been investigated intensively, the particular events following leukocyte adhesion and leading to the actual transendothelial migration process remain largely unknown. To characterize intraendothelial signals elicited by leukocyte adhesion and functionally required for their transmigration, we recorded endothelial free cytosolic intracellular Ca(2+)levels ([Ca(2+)]i) during the course of leukocyte adhesion. We show that monocyte and granulocyte adhesion induced Ca(2+)transients in either untreated or TNF-alpha-stimulated microvascular endothelial cells (HMEC-1). The functional significance of these [Ca(2+)]i rises was demonstrated by treating filter-grown endothelial monolayers with BAPTA/AM. This in traendothelial Ca(2+)chelation left monocyte adhesion basically unaffected, but caused a significant and dose-dependent reduction of the transendothelial migration of monocytes. Granulocyte diapedesis, on the other hand, was hardly modified. Thapsigargin-treatment of endothelial cells almost completely inhibited the transmigration of monocytes suggesting that the necessary Ca(2+)transients depended on a release from intracellular Ca(2+)stores. Our results thus show that the transmigration of monocytes through endothelial monolayers of microvascular origin is favoured by an increase of the intraendothelial [Ca(2+)]i induced by leukocyte adhesion to the endothelial cells.     

Binding site for p120/delta-catenin is not required for Drosophila E-cadherin function in vivo

Homophilic cell adhesion mediated by classical cadherins is important for many developmental processes. Proteins that interact with the cytoplasmic domain of cadherin, in particular the catenins, are thought to regulate the strength and possibly the dynamics of adhesion. beta-catenin links cadherin to the actin cytoskeleton via alpha-catenin. The role of p120/delta-catenin proteins in regulating cadherin function is less clear. Both beta-catenin and p120/delta-catenin are conserved in Drosophila. Here, we address the importance of cadherin-catenin interactions in vivo, using mutant variants of Drosophila epithelial cadherin (DE-cadherin) that are selectively defective in p120ctn (DE-cadherin-AAA) or beta-catenin-armadillo (DE-cadherin-Delta beta) interactions. We have analyzed the ability of these proteins to substitute for endogenous DE-cadherin activity in multiple cadherin-dependent processes during Drosophila development and oogenesis; epithelial integrity, follicle cell sorting, oocyte positioning, as well as the dynamic adhesion required for border cell migration. As expected, DE-cadherin-Delta beta did not substitute for DE-cadherin in these processes, although it retained some residual activity. Surprisingly, DE-cadherin-AAA was able to substitute for the wild-type protein in all contexts with no detectable perturbations. Thus, interaction with p120/delta-catenin does not appear to be required for DE-cadherin function in vivo.     

Claudin-4 is required for AMPK-modulated paraceliular permeability in submandibular gland ceils

Tight junction plays an important rote in mediating paraceUular permeability in epithelia. We previously found that activation of AMP- activated protein kinase （AMPK） increased saliva secretion by modulating paraceUular permeability in submandibular glands. However, the molecular mechanisms underlying AMPK-modulated paraceUular permeability are unknown. In this study, we found that AICAR, an AMPK agonist, increased saliva secretion in the isolated rat submandibular glands, decreased transepithelial electrical resistance （TER）, and increased 4 kDa FITC-dextran flux in cultured SMG-C6 cells. AICAR also induced redistribution of tight junction protein claudin-4, but not claudin-1, claudin-3, occtudin, or ZO-1, from the cytoplasm to the membrane. Moreover, knockdown of claudin-4 by shRNA suppressed while claudin-4 re-expression restored the TER and 4 kDa FITC-dextran flux responses to AICAR. Additionally, AICAR increased ERK1/2 phosphorylation, and inhibition of ERK1/2 by U0126, an ERK1/2 kinase inhibitor, or by siRNA decreased AICAR-induced TER responses. AICAR induced the serine S199 phosphorylation of claudin-4 and enhanced the inter- action of claudin-4 and occludin. Furthermore, pretreatment with U0126 significantly suppressed AMPK-modulated phosphorytation, redistribution, and interaction with occludin of claudin-4. Taken together, these results indicated that claudin-4 played a crucial role in AMPK-rnodutated paraceUular permeability and ERK1/2 was required in AMPK-modulated tight junction barrier function in subman- dibular gland.     

The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres

In vertebrates, centromeres lack defined sequences and are thought to be propagated by epigenetic mechanisms involving the incorporation of specialized nucleosomes containing the histone H3 variant centromere protein (CENP)-A. However, the precise mechanisms that target CENP-A to centromeres remain poorly understood. Here, we isolated a multi-subunit complex, which includes the established inner kinetochore components CENP-H and CENP-I, and nine other proteins, from both human and chicken cells. Our analysis of these proteins demonstrates that the CENP-H-I complex can be divided into three functional sub-complexes, each of which is required for faithful chromosome segregation. Interestingly, newly expressed CENP-A is not efficiently incorporated into centromeres in knockout mutants of a subclass of CENP-H-I complex proteins, indicating that the CENP-H-I complex may function, in part, as a marker directing CENP-A deposition to centromeres.     

Formin-2 is required for spindle migration and for the late steps of cytokinesis in mouse oocytes

Female meiotic divisions in higher organisms are asymmetric and lead to the formation of a large oocyte and small polar bodies. These asymmetric divisions are due to eccentric spindle positioning which, in the mouse, requires actin filaments. Recently Formin-2, a straight actin filaments nucleator, has been proposed to control spindle positioning, chromosome segregation as well as first polar body extrusion in mouse oocytes. We reexamine here the possible role of Formin-2 during mouse meiotic maturation by live videomicroscopy. We show that Formin-2 controls first meiotic spindle migration to the cortex but not chromosome congression or segregation. We also show that the lack of first polar body extrusion in fmn2(-/-) oocytes is not due to a lack of cortical differentiation or central spindle formation but to a defect in the late steps of cytokinesis. Indeed, Survivin, a component of the passenger protein complex, is correctly localized on the central spindle at anaphase in fmn2(-/-) oocytes. We show here that attempts of cytokinesis in these oocytes abort due to phospho-myosin II mislocalization.     

Dynamics between actin and the VE-cadherin/catenin complex: Novel aspects of the ARP2/3 complex in regulation of endothelial junctions

Endothelial adherens junctions are critical for physiological and pathological processes such as differentiation, maintenance of entire monolayer integrity, and the remodeling. The endothelial-specific VE-cadherin/catenin complex provides the backbone of adherens junctions and acts in close interaction with actin filaments and actin/myosin-mediated contractility to fulfill the junction demands. The functional connection between the cadherin/catenin complex and actin filaments might be either directly through α-catenins, or indirectly e.g., via linker proteins such as vinculin, p120ctn, α-actinin, or EPLIN. However, both junction integrity and dynamic remodeling have to be contemporarily coordinated. The actin-related protein complex ARP2/3 and its activating molecules, such as N-WASP and WAVE, have been shown to regulate the lammellipodia-mediated formation of cell junctions in both epithelium and endothelium. Recent reports now demonstrate a novel aspect of the ARP2/3 complex and the nucleating-promoting factors in the maintenance of endothelial barrier function and junction remodeling of established endothelial cell junctions. Those mechanisms open novel possibilities; not only in fulfilling physiological demands but obtained information may be of critical importance in pathologies such as wound healing, angiogenesis, inflammation, and cell diapedesis.     

Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo

Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2′-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration.     

Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo

Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2′-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration.     

The endothelin receptor-B is required for the migration of neural crest-derived melanocyte and enteric neuron precursors

Mutations in the genes encoding endothelin receptor-B (Ednrb) and its ligand endothelin-3 (Edn3) affect the development of two neural crest-derived cell types, melanocytes and enteric neurons. EDNRB signaling is exclusively required between E10.5 and E12.5 during the migratory phase of melanoblast and enteric neuroblast development. To determine the fate of Ednrb-expressing cells during this critical period, we generated a strain of mice with the bacterial beta-galactosidase (lacZ) gene inserted downstream of the endogenous Ednrb promoter. The expression of the lacZ gene was detected in melanoblasts and precursors of the enteric neuron system (ENS), as well as other neural crest cells and nonneural crest-derived lineages. By comparing Ednrb(lacZ)/+ and Ednrb(lacZ)/Ednrb(lacZ) embryos, we determined that the Ednrb pathway is not required for the initial specification and dispersal of melanoblasts and ENS precursors from the neural crest progenitors. Rather, the EDNRB-mediated signaling is required for the terminal migration of melanoblasts and ENS precursors, and this pathway is not required for the survival of the migratory cells.     

MCC, a new interacting protein for Scrib, is required for cell migration in epithelial cells

To further characterize the molecular events supporting the tumor suppressor activity of Scrib in mammals, we aim to identify new binding partners. We isolated MCC, a recently identified binding partner for β-catenin, as a new interacting protein for Scrib. MCC interacts with both Scrib and the NHERF1/NHERF2/Ezrin complex in a PDZ-dependent manner. In T47D cells, MCC and Scrib proteins colocalize at the cell membrane and reduced expression of MCC results in impaired cell migration. By contrast to Scrib, MCC inhibits cell directed migration independently of Rac1, Cdc42 and PAK activation. Altogether, these results identify MCC as a potential scaffold protein regulating cell movement and able to bind Scrib, β-catenin and NHERF1/2.

Structured summary

MINT-7211022: SCRIB (uniprotkb:Q14160) and MCC (uniprotkb:P23508) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7210609: SCRIB (uniprotkb:Q14160) physically interacts (MI:0915) with MCC (uniprotkb:P23508) by two hybrid (MI:0018)MINT-7210759, MINT-7210792: SCRIB (uniprotkb:Q14160) physically interacts (MI:0914) with PIX beta (uniprotkb:Q14155) by pull down (MI:0096)MINT-7210883, MINT-7210820: SCRIB (uniprotkb:Q14160) physically interacts (MI:0914) with MCC (uniprotkb:P23508) by anti bait coimmunoprecipitation (MI:0006)MINT-7210634, MINT-7210690, MINT-7210731: SCRIB (uniprotkb:Q14160) physically interacts (MI:0914) with MCC (uniprotkb:P23508) by pull down (MI:0096)MINT-7211267: E6 (uniprotkb:P06463) physically interacts (MI:0915) with SCRIB (uniprotkb:Q14160), SNX27 (uniprotkb:Q96L92), UTRN (uniprotkb:P46939), CASK (uniprotkb:O14936), DMD (uniprotkb:P11532) and Dlg (uniprotkb:Q12959) by pull down (MI:0096)MINT-7211237: MCC (uniprotkb:P23508) physically interacts (MI:0915) with SCRIB (uniprotkb:Q14160), EZR (uniprotkb:P15311), SNX27 (uniprotkb:Q96L92), NHERF1 (uniprotkb:O14745) and NHERF2 (uniprotkb:Q15599) by pull down (MI:0096)     

The nucleoporin Nup153 maintains nuclear envelope architecture and is required for cell migration in tumor cells

Nucleoporin 153 (Nup153), a component of the nuclear pore complex (NPC), has been implicated in the interaction of the NPC with the nuclear lamina. Here we show that depletion of Nup153 by RNAi results in alteration of the organization of the nuclear lamina and the nuclear lamin-binding protein Sun1. More striking, Nup153 depletion induces a dramatic cytoskeletal rearrangement that impairs cell migration in human breast carcinoma cells. Our results point to a very prominent role of Nup153 in connection to cell motility that could be exploited in order to develop novel anti-cancer therapy.

Structured summary

MINT-7893777: Lamin-A/C (uniprotkb:P02545) and NUP153 (uniprotkb:P49790) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7893761: sun1 (uniprotkb:Q9D666) and Lamin-A/C (uniprotkb:P02545) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

Human Cytomegalovirus (HCMV), an ubiquitous β-herpesvirus, is a significant pathogen that causes medically severe diseases in immunocompromised individuals and in congenitally infected neonates. RhoB belongs to the family of Rho GTPases, which regulates diverse cellular processes. Rho proteins are implicated in the entry and egress from the host cell of mainly α- and γ-herpesviruses, whereas β-herpesviruses are the least studied in this regard. Here, we studied the role of RhoB GTPase during HCMV lytic infection. Microscopy analysis, both in fixed and live infected cells showed that RhoB was translocated to the assembly complex/compartment (AC) of HCMV, a cytoplasmic zone in infected cells where many viral structural proteins are known to accumulate and assembly of new virions takes place. Furthermore, RhoB was localized at the AC even when the expression of the late HCMV AC proteins was inhibited. At the very late stages of infection, cellular projections were formed containing RhoB and HCMV virions, potentially contributing to the successful viral spread. Interestingly, the knockdown of RhoB in HCMV-infected cells resulted in a significant reduction of the virus titer and could also affect the accumulation of AC viral proteins at this subcellular compartment. RhoB knockdown also affected actin fibers'' structure. Actin reorganization was observed at late stages of infection originating from the viral AC and surrounding the cellular projections, implying a potential interplay between RhoB and actin during HCMV assembly and egress. In conclusion, our results demonstrate for the first time that RhoB is a constituent of the viral AC and is required for HCMV productive infection.