Hemidesmosomes and Focal Adhesions Treadmill as Separate but Linked Entities during Keratinocyte Migration

Pora, Anne; Yoon, Sungjun; Windoffer, Reinhard; Leube, Rudolf E.

doi:10.1016/j.jid.2019.03.1139

Cited by 28 publications

(34 citation statements)

References 57 publications

(69 reference statements)

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“…On the other hand, keratin acts as a brake on the actin cytoskeleton presumably responding to pro-migratory cues with a slight delay and by providing a counterbalance to the propulsive actomyosin contractility (for vimentin see 41 ). The latter function is supported by the separate anchorage of the actin and keratin system to focal adhesions and hemidesmosomes 29,[42][43][44] , respectively, and the localization of both systems in separate layers with only limited connectivity (this work; cf. see also 8,45,46 ).…”

Section: Discussionmentioning

confidence: 91%

“…cell culture conditions. Normal human epidermal keratinocytes (nHEKs) derived from neonatal foreskin were purchased from Cell Systems and handled as previously described 48 . Briefly, nHEKs were grown in Dermalife K Medium Complete without TGFα (Cell Systems) in the presence of penicillin-streptomycin (100 µg.…”

Section: Methodsmentioning

confidence: 99%

“…Immunofluorescence labeling was carried out using standard protocols (cf. 48 ). Fixation was performed without any washing steps in order to avoid cell retraction.…”

Section: Methodsmentioning

confidence: 99%

“…transfections and constructs. Cells were transiently transfected one day after seeding with transit-keratinocyte transfection reagent according to the protocol provided by the manufacturer (Mirus Bio LLC) as described previously 48 . A total amount of 2.5 µg DNA was mixed with 3.75 µL reagent and 250 µL medium for each 35 mm-diameter dish.…”

Section: Methodsmentioning

confidence: 99%

“…imaging conditions. As described previously 48 , structured illumination fluorescence microscopy was performed with an ApoTome.2.microscope (Zeiss) equipped with an oil immersion objective 63x (N.A. 1.4, DIC, Plan apochromat).…”

Section: Methodsmentioning

confidence: 99%

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Regulation of keratin network dynamics by the mechanical properties of the environment in migrating cells

Pora

Yoon

Dreissen

et al. 2020

Sci Rep

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Keratin intermediate filaments provide mechanical resilience for epithelia. They are nevertheless highly dynamic and turn over continuously, even in sessile keratinocytes. the aim of this study was to characterize and understand how the dynamic behavior of the keratin cytoskeleton is integrated in migrating cells. By imaging human primary keratinocytes producing fluorescent reporters and by using standardized image analysis we detect inward-directed keratin flow with highest rates in the cell periphery. The keratin flow correlates with speed and trajectory of migration. Changes in fibronectincoating density and substrate stiffness induces concordant changes in migration speed and keratin flow. When keratinocytes are pseudo-confined on stripes, migration speed and keratin flow are reduced affecting the latter disproportionately. The regulation of keratin flow is linked to the regulation of actin flow. Local speed and direction of keratin and actin flow are very similar in migrating keratinocytes with keratin flow lagging behind actin flow. Conversely, reduced actin flow in areas of high keratin density indicates an inhibitory function of keratins on actin dynamics. together, we propose that keratins enhance persistence of migration by directing actin dynamics and that the interplay of keratin and actin dynamics is modulated by matrix adhesions.Cell migration is a highly complex process with relevance for physiological and pathological situations such as embryogenesis, wound healing and metastasis 1 . It is induced by chemical and biophysical cues. The cytoskeleton is at the core of generating effective locomotion by enabling successive steps of protrusion at the cell front and by facilitating the contractile events at the cell rear in parallel with the regulation of cell-matrix adhesions 2 . The cytoskeleton is composed of three major components, namely actin filaments, microtubules and intermediate filaments. The role of actin filaments and microtubules in cell migration has been extensively studied. In contrast, the contribution of intermediate filaments and especially of epithelial keratin intermediate filaments is still poorly understood 3 .Keratin filaments are the main class of cytoplasmic intermediate filaments in epithelial cells. They belong to a large multigene family encoded by more than 50 genes. Every keratin filament contains equal amounts of type I (acidic) and type II (basic) monomers. Obligatory heterodimers assemble in an antiparallel manner into non-polar tetramers, which further associate laterally and longitudinally to eventually generate 8-12 nm filaments 4-6 .The effect of keratin expression on migration depends on the keratin isoform, the cell type and the environment 3,7,8 . For example, the knockdown of K8/K18 reduces the invasion capacity of squamous cancer cells 9 but increases collective cell migration of cancer cells 10 . Depletion of the entire type II keratin gene cluster in mouse keratinocytes prevents keratin filament assembly and induces an increase in migration speed and invasiv...

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Section: Discussionmentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

“…Immunofluorescence labeling was carried out using standard protocols (cf. 48 ). Fixation was performed without any washing steps in order to avoid cell retraction.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Regulation of keratin network dynamics by the mechanical properties of the environment in migrating cells

Pora

Yoon

Dreissen

et al. 2020

Sci Rep

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Implant surface physicochemistry affects keratinocyte hemidesmosome formation

Raptopoulos

Fischer

Aparicio

2023

J Biomedical Materials Res

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Previous studies have shown hydrophilic/hydrophobic implant surfaces stimulate/hinder osseointegration. An analogous concept was applied here using common biological functional groups on a model surface to promote oral keratinocytes (OKs) proliferation and hemidesmosomes (HD) to extend implant lifespans through increased soft tissue attachment. However, it is unclear what physicochemistry stimulates HDs. Thus, common biological functional groups (NH2, OH, and CH3) were functionalized on glass using silanization. Non‐functionalized plasma‐cleaned glass and H silanization were controls. Surface modifications were confirmed with X‐ray photoelectron spectroscopy and water contact angle. The amount of bovine serum albumin (BSA) and fibrinogen, and BSA thickness, were assessed to understand how adsorbed protein properties were influenced by physicochemistry and may influence HDs. OKs proliferation was measured, and HDs were quantified with immunofluorescence for collagen XVII and integrin β4. Plasma‐cleaned surfaces were the most hydrophilic group overall, while CH3 was the most hydrophobic and OH was the most hydrophilic among functionalized groups. Modification with the OH chemical group showed the highest OKs proliferation and HD expression. The OKs response on OH surfaces appeared to not correlate to the amount or thickness of adsorbed model proteins. These results reveal relevant surface physicochemical features to favor HDs and improve implant soft tissue attachment.

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Protein Ligand Nanopattern Size Selects for Cellular Adhesion via Hemidesmosomes over Focal Adhesions

et al. 2022

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Hemidesmosomes (HDs) are multiprotein complexes that firmly anchor epidermal cells to the basement membrane of skin through the interconnection of the cytoplasmic intermediate filaments with extracellular laminin 332 (Ln332). Considerably less attention has been paid to HDs compared to focal complexes/focal adhesions (FC/FAs) in mechanistic single‐cell structures due to the lack of suitable in vitro model systems. Here nanopatterns of Ln332 (100–1000 nm) are created to direct and study the formation of HD in adherent HaCaT cells. It is observed that HaCaT cells at Ln 332 nanopatterns adhere via hemidesmosomes, in stark contrast to cells at homogeneous Ln332 surfaces that adhere via FC/FAs. Clustering of α6 integrin is observed at nanopatterned Ln332 of 300 nm patches and larger. Cells at 500 nm diameter patterns show strong colocalization of α6 integrin with ColXVII or pan‐cytokeratin compared to 300 nm/1000 nm indicating a threshold for HD initiation >100 nm but a pattern size selection for maturation of HDs. It is demonstrated that the pattern of Ln332 can determine the cellular selection of adhesion types with a size‐dependent initiation and maturation of HDs. The protein nanopatterning approach that is presented provides a new in vitro route to study the role of HDs in cell signaling and function.

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Hemidesmosomes and Focal Adhesions Treadmill as Separate but Linked Entities during Keratinocyte Migration

Cited by 28 publications

References 57 publications

Regulation of keratin network dynamics by the mechanical properties of the environment in migrating cells

Regulation of keratin network dynamics by the mechanical properties of the environment in migrating cells

Implant surface physicochemistry affects keratinocyte hemidesmosome formation

Protein Ligand Nanopattern Size Selects for Cellular Adhesion via Hemidesmosomes over Focal Adhesions

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