More than 80% of Earth’s surface is exposed periodically or continuously to temperatures below 5 °C. Organisms that can live in these areas are called psychrophilic or psychrotolerant. They have evolved many adaptations that allow them to survive low temperatures. One of the most interesting modifications is production of specific substances that prevent living organisms from freezing. Psychrophiles can synthesize special peptides and proteins that modulate the growth of ice crystals and are generally called ice binding proteins (IBPs). Among them, antifreeze proteins (AFPs) inhibit the formation of large ice grains inside the cells that may damage cellular organelles or cause cell death. AFPs, with their unique properties of thermal hysteresis (TH) and ice recrystallization inhibition (IRI), have become one of the promising tools in industrial applications like cryobiology, food storage, and others. Attention of the industry was also caught by another group of IBPs exhibiting a different activity—ice-nucleating proteins (INPs). This review summarizes the current state of art and possible utilizations of the large group of IBPs. Current knowledge about living organisms states that there is no place on the surface of the Earth, or in the outer crust , ocean cCold-adapted microorganisms can be divided into two groups: obligate and facultative psychrophiles. Obligate psychrophiles prefer the temperature below 20 °C; their optimum growth is at ~15 °C. These organisms are found in polar regions, mountains, glaciers, and the deep sea. Facultative psychrophiles are organisms that have an optimal growth temperature above 20 °C, but can survive below 0 °C. They live in environments with periodic and seasonal temperature fluctuations. Currently, the minimum temperature for enabling reproduction is considered −12 °C and at −20 °C metabolism is stopped . Water aThe discovery of ice binding proteins (IBPs) synthesized by cold-adapted microorganisms as a strategy of overcoming low temperatures resulted in an increasing interest in their specificity. Their properties such as lowering the freezing point of water or protection from recrystallization during storage opened up a prospect of IBPs becoming useful tools in commercial applications. Special attention is paid to antifreeze proteins (AFPs), which were found to be beneficial biomolecules for the food industry , agriculThis review describes the biodiversity of IBPs in terms of their structures, roles, sources of origin, and mechanisms of action. The main focus of the article is concentrated on the already existing and potential industrial applications of this fascinating group of biomolecules.Water is an essential molecule for life, but it can also lead to cell death. Freezing is lethal for most organisms. The formation of ice crystals can damage cell membranes, which results in cell disruption. Adapting to life in harsh conditions, psychrophiles have developed a number of strategies that allow them to survive. The strategies can range from behavioral  to physiological including freezing avoiding (FA) by supercooling and freezing tolerance (FT) . FA straThese two survival strategies are realized by the synthesis of specific salts, saccharides, peptides, or proteins, IBPs, whose main role is to depress the freezing temperature and affect the shape and size of the ice crystals. The definition of IBPs is broad  and inclIncreasing the tolerance to freezing is based on two activities of AFPs: thermal hysteresis (TH) and ice recrystallization inhibition (IRI). TH causes the decrease of the freezing point of body fluid and was first described by DeVries et al. in 1969  in polarMarinomonas primoryensis) maintain access to oxygen and nutrients by binding to the ice surface through ice-binding domains of large Ca2+-dependent adhesion proteins  [M. primoryensis remain in the upper reaches of the ice-covered lake where oxygen, nutrients, and light are most abundant [Pseudomonas syringae under conditions from −5 to −12 °C, increase the tolerance to freezing, e.g., by forming ice crystals outside the cell, thereby using the heat generated as a heat source. Furthermore, INPs can cause injuries in the epithelium of fruits and vegetables to give bacteria the access to nutrients [Despite the abovementioned activities of AFPs in the cell, some microorganisms also synthesize secreted AFPs to have a constant access to nutrition and water. Bacteria, yeast, fungi, and algae may live and grow in water microchannels in large sea ice created by extracellular IBPs C. Some A1.5 MDa) ,17,18. MThe first discovery of AFPs was by DeVries et al. over 50 years ago in Antarctic notothenioid fishes . They wePseudopleuronectes americanus) fish AFPs to 1.5 MDa in Antarctic Gram-negative bacterium, Marinomonas primoryensis [Colwellia and yeast Leucosporidium , and non–fish origins that include microbial proteins  and proteins isolated from insects and plants. Characteristic features of the particular groups are described below and summarized in N–acetylgalactosamine) at each hydroxyl group of the side chains of threonines. The AFGP gene derives from the recruitment and iteration of a small region spanning the boundary between the first intron and second exon of the trypsinogen gene. This new segment was expanded and then iteratively duplicated to produce 41 tandemly repeated segments [AFPG is synthesized by Antarctic notothenioids and northern cods and characterized by small molecular weight ranging from 3 to 33 kDa . AFPGs wsegments . Due to segments .Type I AFPs were isolated from right-eyed flounders, sculpins, and Alaskan plaices. These proteins have a small mass ranging from 3 to 4.5 kDa, with the exception of AFP “Maxi” . Their molecular weight ranges from 11 to 24 kDa .Type III AFPs are synthesized by eel pout, ocean pout, and wolfish. They are characterized by a globular shape, a low molecular weight of about 6–7 kDa, and no repeatability in the amino-acid sequences that do not contain cysteine residues. They can be divided into two subgroups: quaternary amino-ethyl (QAE) and sephadex-binding (SP) isoforms, based on sequence similarity and isoelectric point .Secale cereale) [Picea sp.) [Lolium perenne LpAFP) [Pseudomonas sp., which in this way obtain the entrance to the intracellular nutrients [Like fish, plants also synthesize AFPs to protect against freezing damage associated with ice crystal formation in the apoplasts. Plant AFPs have lower TH activity at the expense of IRI activity ,43. Duricereale) . This AFcereale) . Most ofcea sp.) . Interese LpAFP) . Furthere LpAFP) . Most ofe LpAFP) . They thutrients .Lepidoptera), which belong to different insect families [Tenebrio molitor beetle by Ramsay in 1971, during his study on the rectal complex [Insect proteins are so far the most active characterized AFPs . The TH families . AFPs befamilies . The fir complex . At that complex .The discovery of fish AFPs was followed by extensive search for other sources of these specific molecules motivated by the cost of purification and problems with large scale production of the recombinant fish proteins . It turnGlaciozyma sp. AY30, Glaciozyma antarctica) and three snow mold fungi  are able to synthesize these proteins at subzero temperatures (Glaciozyma sp. AY30 (formerly Leucosporidium) and was isolated from ice core sample of a freshwater pond near the Dasan station, Ny–Ålesund, Svalbard archipelago, Norway [E. coli and P. pastoris in the pilot scale (700 L) and the obtained yields were at 300 mg/L [Glaciozyma antarctica PI12. This strain produces two isoforms of antifreeze proteins: Afp1 with molecular mass of ~18 kDa [Typhula iskariensis and Afp4 shows 93% similarity with LeIBP (Glaciozyma sp. AY30). Both Afp1 and Afp4 display the activity of TH and IRI. In 2018, Villarreall et al. demonstrated that three other species of Antarctic yeast  show antifreeze activity, but these proteins were not characterized [It has been demonstrated that only two yeast strains  [Antarctomyces psychrotrophicus (AnpAFP) is an extracellular protein of approximately 28 kDa [B: 3VN3)  and a hyB: 3VN3) . The molB: 3VN3) ,66. The y 28 kDa . AnpAFP y 28 kDa .Collwellia strain SLW05 (ColAFP) [Marinomonas primoryensis produces a > 1 MDa protein with calcium-dependent activity (Flavobacterium frigoris PS1 (FfIBP) synthesize the AFP, which has 56% structural similarity to AFP derived from Leucosporidium yeast (LeIBP), while having 10 times more TH activity than LeIBP [In recent years, many AFPs have been discovered from bacteria found in cold habitats . Unfortu(ColAFP) . In 2014(ColAFP) . Based o(ColAFP) . Marinomactivity E. The moactivity J. Recentan LeIBP . Higher an LeIBP . The FfIan LeIBP .Pseudomonas syringae) [Unlike AFPs, INPs are large, hydrophilic, and multimeric proteins with subunits from 120 to 150 kDa in weight . Most ofyringae) .Based on the models of 3D structures of these proteins, INPs consist of three domains; the first—N–terminal, with a role anchoring the protein in the cell membrane, the second—the region of 16 amino-acid tandem repeats , third domain—C–terminal, which function remains unknown . These pPseudoaletoromonas) and yeast (Glaciozyma sp. AY30) additionally produce enhancer molecules such as polysaccharides , amino acids , or salts , which increase the TH of AFPs [AFPs exhibit two activities that are related to their affinity to ice: TH and IRI B–D. In n of AFPs ,83,84.Ice recrystallization can be described as the process of creation the larger ice crystals at the loss of smaller ones via the Kelvin effect . The secRhagium inquisitor, RiAFP) was crystallized as a dimer which, however, was not stable in solution because of higher shape complementarity for RiAFP and ice than for two subunits of RiAFP’s crystallographic dimer [Most of AFPs have in their structure internal asparagine ladders, an external α-helix, or an extensive network of hydrogen bonds in the protein core, which keeps them rigid . Anotheric dimer .Marinomonas primoryensis), and a hyperactive insect AFP (beetle Microdera punctipennsis dzungarica) [According to the all of the above, three hypotheses of possible ice binding mechanisms of AFPs could be taken into consideration. The first hypothesis about IBPs binding to ice surfaces arose in 1977 based on research of DeVries and Raymond on fish AFPs type I and AFGP . It was ngarica) . Moleculngarica) .It is also worth adding that in the case of AFGPs, the disaccharide residue  plays an important role in binding to ice . MeisterIn the group of IBPs, in addition to proteins that inhibit the growth of ice crystals, there are proteins that have the ability to initiate ice crystallization at temperatures from 0 to −2 °C . INPs arThe mechanisms that use INPs to create ice crystals are still not solved in contrast to the effect of IBPs on inhibiting the growth of ice crystals which is now well understood . NumerouThe discovery of AFPs resulted in an increasing interest in their applications, which are concentrated in the areas of food technology, agriculture, cryobiology, and material technology .One of the most important industries where AFPs can be applied is food manufacturing. AFPs used as a food supplement improve the quality of stored frozen products, e.g., by keeping a smooth texture without the ice grains of ice cream or by increasing the time of storage of meat, fish, fruits, dough, and vegetables. Many patents can be found that describe AFPs in food storage ,103.The most common problem in the production of ice cream is the ice recrystallization caused by temperature differences during product storage. Small ice crystals melt and turn into larger ones when the temperature decreases. Application of AFPs changes the texture and organoleptic characteristics of ice cream and improves them.AFPs type III derived from psychrophilic fish were allowed for consumption in ice cream in 2008 by the European Food Safety Authority in Europe and in 2013 by the Food and Drug Administration in USA ,104. AFPAvena sativa). The research comprised purification and identification of AFPs, called AsAFP, and analysis of TH activity. After the discovery that purification decreases TH activity, it was assumed that the reason was washing out low mass substances like activators, sugars, and antibodies that could enhance the positive effect of AFPs. IRI activity of AsAFP in ice cream was triggered by applying 0.1% (w/w) AsAFP or BSA to the samples, fast cooling and heating, fluctuating temperature, and observing the effects using a microscope. After violent cooling, there was no spectacular change in ice crystal size but during temperature fluctuation, the difference between the sample with AsAFP and with BSA was observed. There were smaller ice crystals in ice cream with addition of AsAFP than in ice cream with addition of BSA. Another measured parameter was melting ratio which is affected by many factors like ice cream composition, ice crystal size, consistency [Zhang et al., 2016 used AFPs isolated from cold-acclimated oat  of cultivar “Visello” on texture and microtexture of low-fat ice cream. The obtained extract contained 16.4% (w/w) total protein, soluble polysaccharides, and salts. The assay of AFP activity was also done using recombinant AFP with C-terminal residues YPPA mutated to YAAKDEL to improve protein solubility. The expression was performed in E. coli BL 21 strain. Two sets of samples were prepared—one of them was enriched with winter rye extract containing up to 400 mg/L lyophilized extract dry weight total protein and the second one was pricked with E. coli extract containing up to 35 mg/L IBPs. The ice cream was frozen to −5 °C then hardened to −40 °C for 24 h. After that samples were stored in −18 °C. Another study that was carried out on the ice cream utilized the measurement of IRI activity by using a modified sucrose sandwich assay method [E. coli it amounted to 0.17 mg/mL and 0.10 µg/mL, respectively. There is a hypothesis that recombinant AFP’s IRI activity is higher than observed by other groups because in this experiment ice cream mix was used. Microstructure of ice cream was observed by using polarized light microscopy and in comparison with the control sample—pure ice cream—ice crystals in ice cream with addition of IBPs were tinier as expected but also aggregated into small groups. The occurrence of these agglomerates would suggest that new structures from ice crystals formed by AFPs arise. In order to check the sweetness, roughness, and friability of the samples, a panel comprising seven trained sensory assessors was conducted. Assessors could check the mouthfeel only of the ice cream with winter rye extract and control group. Furthermore, the hardness of both sets of samples and control group was tested by applying the method proposed by Valera et al., 2014 [Kaleda et al., 2018 tested the impact of AFPs extracted from winter rye leaves  which constituted 15.4% (w/w) of the added concentrated carrot protein (CCP). It was proven that in comparison with other cryoprotectants—BSA  and SPI (soy protein isolate), samples with DcAFP after baking were characterized by a greater volume of loaf, deeper color of crust (as a result of the Maillard reaction induced by presence of AFPs), whiter crumb, and softness and smoothness in mouthfeel. Application of DcAFP lowered hardness of the dough because AFPs captured water molecules (even they were not frozen on the ice surface), improved fermentation capacity, and lead to forming less ice crystals in comparison with control samples by inducing TH activity. In addition, it was found that application of carrot juice lead to better taste and smell than using a solution of purified AFPs [The positive impact of AFPs on frozen dough was verified in 2007 ,115. Theied AFPs . Similaried AFPs  and barlied AFPs .It was also determined that DcAFP at a concentration 1.29 mg/mL on frozen white salted noodles enhanced the cooking properties and improved noodle texture . Applica6 units per gram of dough were considered. These IBPs were derived from Erwinia herbicola also known as Enterobacter agglomerans [Not only AFPs were investigated to act as protectants during storage of frozen dough but also extracellular INPs at a concentration of 2.4 × 10lomerans . Unlike The biggest problem during meat freezing is the formation of big, needle-shaped ice crystals that damage cell membranes, causing significant water loss. Assays of application of AFPs as cryoprotectants of frozen meat relied on IRI activity, which resulted in small ice grain formation, which prevented destruction of the cells. One of the preliminary ideas was to soak the samples of meat in the AFP solution before freezing. During this experiment, the samples were soaking too long and spoiled. In order to avoid similar situations, injection the AFP solution to living lamb before slaughter was suggested . The conRecently used cryoprotectants, 4% sucrose or sorbitol, cause sweetness of meat, which is an adverse effect because of taste and medical reasons. That is another superiority of AFPs which lack flavor .Until present there was no research on the impact of AFPs of different sources on freezing and storage of fresh fruit or vegetables. The only report was about carrot being preserved by synthetic peptides that have similar properties to IBPs . Cubes oThe next potential and the most promising area of application of AFPs is cryopreservation. Transplantation and cryosurgery require new natural methods that facilitate the storage of tissues and organs at low temperatures. In order to extend the life of cells, or to protect them from damage caused by ice crystals, two types of chemical substances were used: cell penetrating cryoprotectants (ethylene glycol (EG), dimethyl sulfoxide (DMSO), and glycerol) and non–penetrating cryoprotectants . Recent studies have shown that AFPs can potentially replace these agents, because they are less toxic and non–penetrating. Moreover, they effectively inhibit the growth of ice crystals at lower concentrations than conventional substances; however, a deeper insight into the mechanism of freezing damage is required.Glaciozyma AY30) appeared in 2011 and related to the positive effect of the addition of this protein on the osmotic resistance, motility, and viability of wild boar sperm during freezing [Glaciozyma AY30 yeast on red blood cells [Glaciozyma sp. AY30. Koh and co–authors used the marine diatom Phaeodactylum tricornutum, which is a model organism and has already been the object of many ecological, physiological, biochemical, and molecular studies [Glaciozyma sp. AY30), FfIBP (Flavobacterium frigoris), and type III AFP (Zoarces americanus) on cryopreservation of ovaries frozen by vitrification, and their subsequent warming and transplantation into mice. The results showed that supplementing AFPs at a concentration of 20 mg/mL in the vitrification solution had a protective effect on the survival of ovarian tissue during cryopreservation and transplantation, and that the most beneficial activity was observed for the LeIBP [The first reports on the use of AFPs as cryoprotectants date from the 1990s when proteins from Antarctic and Arctic fish were investigated . Since tfreezing . Subsequod cells . The add studies . When ce studies . MoreoveAn important application of AFPs in cryopreservation is also hypothermic organ storage . HoweverChoristoneura fumiferana [A. thaliana with perennial ryegrass cv. Caddyshack (Lolium perenne) gene  which can survive the temperatures between 4 and −8 °C [A. thaliana resistance to temperatures from −5 to −8 °C, also using a gene from perennial ryegrass v. Pacific Seed Diploid (Lolium perenne) [Another important branch of industry in which AFPs can be used is agriculture. So far, AFPs produced by plants resistant to temperature fluctuations (survive during extracellular freezing) have been characterized. Plants produce their own AFPs, however, with low TH and IRI activity, which do not completely protect them from the formation of ice crystals, contrary to AFPs in fish or insects. Attempts were made to express these proteins in plants to immunize them for periodic temperature decrease even to −5 °C. First reports on the creation of cold resistant plants come from the 1990s. A trial of AFP type I protein expression from marine fish was made using potatoes, tomatoes, and tobacco ,127,128;miferana . The trand −8 °C . Anotherperenne) . The obtperenne) . UnprediExcept for the food industry, agriculture and cryopreservation, the areas of potential application of IBPs are still growing, e.g., in sectors of industry such as: material technology, the fuel industry, and weather control.The ability of AFPs to change the morphology of ice crystals brought the idea of introduction of these proteins to a wide range of materials, including macroporous ceramic materials, polymers, and complex composites for the production of catalysts, absorbents, tissue engineering scaffolds, transport systems, and strong materials of low weight . One exaChaetoceros neogracile (Cn-AFP), to prevent the formation of ice crystals on refrigeration surfaces. They created a recombinant fusion protein consisting of AFP (Cn-AFP) and Al–binding peptides. After 3 h of freezing, no ice crystals were observed on Al bars coated with AFPs, compared to controls where the frosting was significant. The results of this experience can be used in kinds of metals in aviation or in refrigeration, where the formation of large ice crystals is a crucial parameter.The next interesting use of AFPs is applying them as potential anti-icing substances . Gwak etFinally, Wilkins et al., 2019 have recently demonstrated that AFPs can be conjugated onto polymer-stabilized gold nanoparticles. This may lead to the application of AFPs organized into complex assemblies in colloid science or in basic sciences utilizing the nanoparticle cores for tracking and bioimaging . Moreove®, created from lyophilized, non-viable bacteria from Pseudomonas syringae species [P. syringae work as snow inducers and improve the crystallization process [In contrast, INPs are widely used as factors affecting the process of atmospheric glaciation, as a consequence of precipitation and cloud formation. Currently, these proteins are used for the production of artificial snow. There is a commercial product called Snomax species . Protein process . Apart f process . Other s process . The lev process ,141.Psychrophiles represent a significant group of organisms; hence they are an interesting object to study in academia and biomedical fields. Much is said about extremophiles in the context of the source of unique biomolecules used in different types of industry. Especially valuable from a technological point of view are the enzymes which due to their numerous structural and kinetics adaptations have already found many applications in food technology, molecular biology, medicine, etc. Furthermore, the new intelligent biopolymers, antibiotics, anti-cancer drugs, and IBPs isolated from cold-loving microorganisms also deserve attention. IBPs recognized as cryoprotectants are becoming increasingly desirable for the agri-food industry, material science, coatings, and biomedical applications. Their potential is certainly much greater, but not yet fully understood, despite the clear progress observed in the last years in the structural and functional characteristics of these biomolecules. However, there are still many unanswered questions related to e.g., the relations between IBP types, their activity, and ice binding mechanisms. Great hopes associated with this are found in the intensive development of genetic engineering, synthetic biology, and bioinformatics. The ultimate goal should be the manufacture of IBPs in high yields at low cost for scientific purposes and successful commercialization. It should be also remembered that control over the nucleation and growth of ice crystals is crucial for the survival of various species, atmospheric glaciation processes, and decisive for the structural integrity and properties of a broad range of water-based materials.


Undaria pinnatifida seaweed species against three starch hydrolyzing enzymes—α-amylase, α-glucosidase, and amyloglucosidase—was investigated. It was demonstrated that while the fucoidan exhibited significant inhibitory effects against all the three starch hydrolases, it is an uncompetitive inhibitor of α-amylase and amyloglucosidase, and is a competitive inhibitor of α-glucosidase. Moreover, it exhibited significantly stronger inhibitory effects against α-glucosidase than α-amylase, thus having the desirable characteristics as an antidiabetic agent.Fucoidan refers to a group of sulfated polysaccharide that is commonly obtained from various species of brown seaweed. Fucoidan has gained increased popularity among researchers in the recent years due to its numerous biological activities, including its inhibitory effects against starch hydrolyzing enzymes such as α-amylase and α-glucosidase. This highlights the potential of fucoidan as an antidiabetic agent in the management and prevention of diabetes mellitus. In this study, the inhibitory effects of fucoidan isolated from the New Zealand  In the ell line . Howeverell line . As suchell line .Ascophyllum nodosum exhibited inhibitory activity against α-amylase and α-glucosidase, while fucoidan isolated from Fucus vesiculosus exhibited inhibitory activity against α-amylase only. Moreover, it has been reported that the starch hydrolase inhibitory activity of fucoidan is also influenced by its sulfate content. U. pinnatifida and chemically modified them to increase the level of sulfate content. They reported that over-sulfation of fucoidan enhanced the inhibition effect on amyloglucosidase but not on α-amylase  and α(1–4) galactose and fucose units, which has been associated with its superior anticancer activity against T-47D breast cancer cell line and SK-MEL-28 melanoma cancer cell lines than fucoidan from other seaweed species  solution with constant stirring. The sodium potassium tartrate solution was heated and maintained at 50–70°C, and care was taken to avoid boiling of the solution. A 96 mM 3,5-dinitrosalicylic acid (DNS) solution was prepared by solubilizing DNS in deionized water. The DNS solution was heated and maintained at 50–70°C. Deionized water was heated to 60°C and the deionized water, sodium potassium tartrate solution, and DNS solution were allowed to mix slowly in the ratio of 3:2:5. The resulting mixture was stirred to ensure complete dissolution. The working color reagent was stored in an amber flask at room temperature for use.Aliquots of fucoidan solution of 20 μl and α-amylase solution of 20 μl were added into a 2 ml Eppendorf tube and incubated at 37°C water bath for 15 min to allow for interactions between the enzyme and fucoidan. The enzyme reaction was initiated by adding 60 μl of starch solution into the Eppendorf tube containing the enzyme-fucoidan reaction mixture. The reaction was allowed to proceed for 5 min at 37°C. Subsequently, 100 μl of DNS color reagent was added into the Eppendorf tube and incubated at 100°C for 15 min to allow for color development. The reaction mixture was then cooled in an ice water bath. Aliquots of 200 μl of reaction mixture were transferred into a 96-well plate and the absorbance of the reaction mixture was read at 540 nm using a microplate reader . A blank was prepared for each fucoidan concentration used by preparing the reaction mixture in the same way and replacing α-amylase and starch solution with SPB. This is to avoid any interference caused by the color of fucoidan. A control sample was prepared by replacing α-amylase with an equivalent volume of SPB.A standard curve of various concentrations of maltose was constructed to quantify the amount of liberated maltose. For each concentration of maltose, 100 μl of maltose solution was allowed to react with 100 μl of DNS color reagent solution. The reaction mixture was heated at 100°C for 15 min and cooled in an ice water bath. Aliquots of 200 μl of reaction mixture were transferred into a 96-well microplate and the absorbance of the reaction mixtures was read at 540 nm using a microplate reader. The standard curve was constructed by plotting a graph of absorbance at 540 nm against concentration of maltose standard. The absorbance of the sample reaction mixtures was then fitted into the standard curve to determine enzyme activity.p-nitrophenol liberated from 4-nitrophenyl α-D- glucopyranoside according to the method by The inhibition activity of fucoidan against α-glucosidase was determined by measuring the amount of 2CO3 solution was added into the Eppendorf tube. Aliquots of 200 μl of reaction mixture were transferred into a 96-well plate and the absorbance of the reaction mixture was read at 405 nm using a microplate reader . A blank was prepared for each fucoidan concentration used by preparing the reaction mixture in the same way and replacing α-glucosidase and 4-nitrophenyl α-D- glucopyranoside solution with SPB. This is to avoid any interference caused by the color of fucoidan. A control sample was prepared by replacing α-glucosidase with an equivalent volume of SPB.Aliquots of fucoidan solution of 120 μl, α-glucosidase solution of 60 μl, and SPB of 300 μl were added into a 2 ml Eppendorf tube and incubated at 37°C water bath for 15 min to allow for interactions between the enzyme and fucoidan. The enzyme reaction was initiated by adding 120 μl of substrate solution into the Eppendorf tube containing the enzyme-fucoidan reaction mixture. The reaction was allowed to proceed for 10 min at 37°C. Subsequently, 300 μl of Nap-nitrophenol was constructed. The absorbance of the sample reaction mixtures was fitted into the standard curve to determine enzyme activity.A standard curve of various concentrations of The inhibition activity of fucoidan against amyloglucosidase was determined by measuring the reducing power of released reducing sugar from soluble starch according to the method by Aliquots of fucoidan solution of 20 μl and amyloglucosidase solution of 20 μl were added into a 2 ml Eppendorf tube and incubated at 37°C water bath for 15 min to allow for interactions between the enzyme and fucoidan. The reaction was initiated by adding 60 μl of starch solution into the Eppendorf tube and the reaction was allowed to proceed for 5 min at 37°C. Subsequently, 100 μl of DNS color reagent was added into the Eppendorf tube and incubated at 100°C for 15 min to allow for color development. The reaction mixture was cooled in an ice water bath and 200 μl of reaction mixture was transferred into a 96-well plate. The absorbance of the reaction mixture was read at 540 nm using a microplate reader . A blank was prepared for each fucoidan concentration used by preparing the reaction mixture in the same way and replacing amyloglucosidase and starch solution with SPB. A control sample was prepared by replacing amyloglucosidase with an equivalent volume of SPB.A standard curve of various concentrations of maltose was constructed to quantify the amount of liberated maltose. The absorbance of the sample reaction mixtures was fitted into the standard curve to determine enzyme activity.MaxV) and the Michaelis-Menten constant (MK).The type of enzyme inhibition was determined graphically using the Michaelis-Menten plot and Lineweaver-burk plot. The data sets obtained from each enzyme inhibition assay were analyzed using the nonlinear regression curve fit in GraphPad Prism software  to determine maximal velocity  between the samples.All the analysis was performed in triplicates. The results are expressed as the mean values of the triplicate measurements together with their standard deviations. Statistical analysis was conducted by one-way ANOVA using SPSS 21 software . via the Michaelis-Menten plot in The inhibition activity of fucoidan against α-amylase was demonstrated in this study MaxV) and Michaelis-Menten constant (MK) values were obtained and tabulated as shown in MaxV and MK decreased significantly with increasing concentration of fucoidan. This is characteristic of an uncompetitive inhibitor  plot as shown in nhibitor . In unconhibitor . This imnhibitor . Therefonhibitor . This wiU. pinnatifida is an inhibitor of α-glucosidase as shown in the Michaelis-Menten plot in Fucoidan from MaxV and MK values were obtained and tabulated as shown in MK decreased significantly with increasing concentration of fucoidan while MaxV did not change significantly with increasing fucoidan concentration. This is characteristic of a competitive inhibitor  plot as shown in nhibitor . In compnhibitor . Thus, bnhibitor .via the Michaelis-Menten plot in U. pinnatifida also exhibited inhibitory effects against amyloglucosidase activity.The inhibition activity of fucoidan against amyloglucosidase was demonstrated in this study MaxV) and Michaelis-Menten constant (MK) values were obtained and tabulated as shown in MaxV and MK decreased significantly with increasing concentration of fucoidan. This further confirms that the fucoidan was an uncompetitive inhibitor of amyloglucosidase  plot as shown in cosidase .Fucoidan extracted from U. pinnatifida has been demonstrated in Inhibition Type of Fucoidan Against α-Amylase, Inhibition Type of Fucoidan Against α-Glucosidase, and Inhibition Type of Fucoidan Against Amyloglucosidase to exhibit inhibitory effects against the three key starch hydrolases—α-amylase, α-glucosidase, and amyloglucosidase. However, the exact mechanisms by which fucoidan inhibited the three different starch hydrolases has yet to be fully elucidated. Moreover, it was observed that fucoidan from U. pinnatifida exhibited differential inhibitory potency against the three different starch hydrolases.F. vesiculosus and A. nodosum. It was reported that fucoidan from A. nodosum exhibited inhibition against α-amylase while fucoidan from F. vesiculosus did not (F. vesiculosus and A. nodosum are largely composed of α-(1–3) linked sulfated L-fucose units in its backbone structure, a low proportion α-(1–4) linked fucose or a repeating α-(1–3) and α-(1–4)-linkage is only found in the backbone structure of fucoidan from A. nodosum  and α(1–4) fucose and galactose units, with a high degree of sulfation  % exhibited significantly inhibitory effects against α-amylase. Thus this suggests that while sulfate content might play a role in α-amylase inhibition, it has to be considered together with other factors or structural features involved in the α-amylase inhibitory activities of sulfated polysaccharides like fucoidan.Moreover, the inhibition effect of fucoidan against α-amylase has been reported to be dependent on its sulfate content . Cho et -amylase . Howevervia its hydrogen scavenging activity, which is similar to the α-glucosidase inhibition mechanism of most polyphenolic compounds . This is similar to the inhibition mechanism of acarbose on α-amylase reported by As reported in this study, fucoidan from U. pinnatifida. Moreover, in the case of amyloglucosidase, the binding of fucoidan to the secondary binding site of enzyme could be facilitated by the electrostatic interactions between the negatively charged sulfate groups of fucoidan and the enzyme’s secondary binding site. This may also account for the enhanced amyloglucosidase inhibitory effects of fucoidan with higher sulfate content reported in the literature (via electrostatic interactions involving sulfate groups of fucoidan, as well as by increasing the viscosity of the reaction medium.A similar mechanism can be proposed for the uncompetitive inhibition of amyloglucosidase by fucoidan from terature . Anotherterature . OverallThe inhibitory effects of fucoidan against α-amylase, α-glucosidase, and amyloglucosidase highlight the potential of fucoidan to be used as an anti-diabetic agent. Currently, there are many commercially available drugs in the market for diabetes mellitus treatment such as acarbose. However, antidiabetic drugs e.g. acarbose have numerous side effects such as abdominal distension, flatulence, meteorism, and diarrhea as a result of the strong inhibitory effects of acarbose against α-amylase . AcarbosOne way to reduce the adverse effects associated with acarbose is to replace it with an inhibitor that is more potent against α-glucosidase than α-amylase . Thus, m50 of fucoidan inhibition against α-glucosidase was significantly lower than those against α-amylase and amyloglucosidase, despite of a higher substrate concentration (50 value for fucoidan inhibition of glucosidase suggests that U. pinnatifida is likely to have more potent inhibitory effects against α-glucosidase than α-amylase. This further expounds the benefits of fucoidan from U. pinnatida in diabetes prevention and management as compared to the popular anti-diabetic drugs such as acarbose. However, further works need to be conducted to validate the different potency of U. pinnatifida fucoidan on α-amylase and α-glucosidase.In this study, it was observed that the ICU. pinnatifida against three key starch hydrolases—α-amylase, α-glucosidase, and amyloglucosidase. Based on the Lineweaver-Burk plots, as well as maximal velocity and Michaelis-Menten constant, it is concluded that fucoidan from U. pinnatifida is an uncompetitive inhibitor of both α-amylase and amyloglucosidase, and a competitive inhibitor of α-glucosidase. In addition, based on the IC50 values, it has been demonstrated that fucoidan from U. pinnatifida is likely to exhibit significantly stronger inhibitory effects against α-glucosidase than α-amylase and amyloglucosidase. As such, fucoidan from U. pinnatifida possesses the desirable attributes as a potential anti-diabetic agent and offers new ideas in diabetes management and prevention.This study demonstrated the inhibitory effects of fucoidan isolated from New Zealand All datasets generated for this study are included in the article/supplementary material.JL and WZ conceived project. HK carried out the experiment. JL provided materials. WZ provided resources. JL and WZ supervised HK. HK analyzed the data and wrote the manuscript. JL and WZ edited the manuscript. All authors have read and agreed to submit this manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ERK5 is a protein kinase that also contains a nuclear localisation signal and a transcriptional transactivation domain. Inhibition of ERK5 has therapeutic potential in cancer and inflammation and this has prompted the development of ERK5 kinase inhibitors (ERK5i). However, few ERK5i programmes have taken account of the ERK5 transactivation domain. We have recently shown that the binding of small molecule ERK5i to the ERK5 kinase domain stimulates nuclear localisation and paradoxical activation of its transactivation domain. Other kinase inhibitors paradoxically activate their intended kinase target, in some cases leading to severe physiological consequences highlighting the importance of mitigating these effects. Here, we review the assays used to monitor ERK5 activities  in cells, the challenges faced in development of small molecule inhibitors to the ERK5 pathway, and classify the molecular mechanisms of paradoxical activation of protein kinases by kinase inhibitors. Protein kinases play key roles in a variety of diseases including cancer and inflammation and have emerged as ‘druggable’ enzymes . By 2019MAPK7 gene [. [The mitogen-activated protein kinase (MAPK) family member, extracellular signal regulated kinase 5  is encoded by the PK7 gene . It is tPK7 gene  and a laPK7 gene   activatPK7 gene , much liPK7 gene ,15 wherePK7 gene ,16,17 . Monitoring the ERK1/2 pathway is made easier by the existence of more than 200 ERK1/2 substrates and interacting proteins .Whilst inhibition of MEK5 is relatively easily assessed in cells by loss of ERK5 activation-loop TEY phosphorylation, the paucity of well-validated ERK5 substrates allowing reliable monitoring of ERK5 activity or inhibition has held back our understanding of ERK5 biology, and ERK5i development and disease positioning. Techniques that have been used to monitor ERK5i-dependent inhibition of ERK5 in cells include:1. KiNativ™. This technique measures binding of small molecules to the kinase active site. It uses biotin-tagged acyl-phosphates of ATP and ADP as probes, which acylate the conserved active site lysines in protein kinases (and other ATP-dependent enzymes). Mass spectrometry is then used to identify streptavidin captured peptides following tryptic digestion. Pre-treatment of cells with and without inhibitors, followed by cell lysis and capture of non-inhibitor-bound ERK5 via the ATP-site probe allows the quantification of target engagement [gagement ,43. This2. ERK5 autophosphorylation assay. MEK5-catalysed phosphorylation of the activation-loop TEY motif activates the ERK5 kinase domain, which subsequently drives autophosphorylation at multiple sites within the C-terminus  and FOS  which bind to the core DNA sequence 5′-TGAG/CTCA-3′. A multimerised AP-1:Luc reporter can be stimulated by ERK5 when it is activated by co-transfected MEK5D (a constitutively active MEK5 mutant). This has been used to assess ERK5 activation and inhibition by ERK5i in cells [de novo expression and proteasomal degradation) and their activity . In terms of dimer composition, some FOS or JUN proteins can also heterodimerise with the ATF transcription factors , MAFs  and the cAMP response element-binding proteins (CREBs). In terms of abundance and activity, the ERK1/2 [in cells . Howevere ERK1/2 ,47,48 ane ERK1/2  pathwayse ERK1/2 ,49.−/− MEFs and this is rescued by re-expression of ERK5 [JUN promoter is regulated by MEF2C, the best characterised substrate of ERK5; ERK5 phosphorylation of MEF2C enhances its transcriptional activity and promotes JUN expression [in vitro [The most likely mechanism by which ERK5 might regulate AP-1 is by promoting JUN expression. JUN expression is decreased in ERK5 of ERK5 . The JUNpression ,51. ERK5pression  and can in vitro , howeverin vitro . Taking 4. MEF2-dependent reporter assays. The best validated ERK5 interacting proteins and substrates are the MEF2A, C and D transcription factors [ factors ,17,54. T factors ,58. The  factors , making A variation of this assay involves fusing the region of MEF2 housing ERK5 phosphorylation sites and the MEF2 TAD to the DNA-binding domain of the yeast transcription factor GAL4 . This METranscription-based readouts of ERK5 activity do not readily differentiate between the contribution of the kinase and TAD activities when a full length construct of (or endogenous) ERK5 is assessed. For example, we have found that a truncation mutant of ERK5 that lacked the TAD could drive MEF2D:GAL4 activity when co-transfected with MEK5D, but also that a kinase dead full-length form of ERK5 (that contains the TAD) could drive MEF2D:GAL4 luciferase activity following ERK5i treatment (see below) . HoweverBy analogy with the RAF–MEK1/2–ERK1/2 pathway, one could conceive of targeting the ERK5 pathway at any step from MEKK2/3 to MEK5 or ERK5 itself . HoweverThe first pathway inhibitors to be reported were the MEK5 inhibitors, BIX02188 and BIX02189. These inhibitors are relatively selective for MEK5, with the strongest off-target effect on Src . They ha25 (XMD17-26) and cpd 26  [26 (XMD17-109) and AX15836, cause a conformational change in the kinase domain which leads to exposure of the C-terminal NLS and paradoxical activation of the ERK5 TAD [The majority of effort has focused on the development of small molecule ERK5i, probably because ERK5 is the ‘effector kinase’ in the pathway. However, ERK5 is proving to be a challenging target. The first ERK5i to be described were XMD8-92 , cpd 25 K5-IN-1) . AlthougK5-IN-1)  — an epiK5-IN-1) . AX15836K5-IN-1) . HeedfulK5-IN-1) ,55,56 an46; this inhibitor is distinct from the XMD8-92 series, has no activity towards BRD4 or LRRK2, is highly selective for ERK5 and is also suitable for use in animal studies [Additional ERK5i in the public domain include JWG-045 and JWG-071, a further development of the XMD8-92 series. Both have selectivity over BRD4 but inhibit LRRK2 and are classed as dual ERK5/LRRK2 inhibitors. The kinase selectivity data for JWG-071 have been published, showing that it only inhibits three other kinases  at low micro molar concentrations. In cell-based assays, an inhibitor that blocks LRRK2 activity  . For a rn et al. . Type IVin cells  but it h46 is a selective ERK5i, with mild paradoxical activation effects, that prevents tumour growth by inhibiting angiogenesis [Some ERK5i have anti-tumour activity . As XMD8ogenesis . These fThe unintended activation of kinase signalling through inhibitor binding to the kinase, termed paradoxical activation, has emerged as a significant challenge to kinase inhibitor development. The first kinase that was reported to be paradoxically activated by selective small molecule inhibitors was PKB. Inhibitor binding promotes PKB membrane localisation, regulatory site phosphorylation (T308 and S473) by PDK1 and mTORC2, and acquisition of a phosphatase-resistant conformation; consequently, when the inhibitor is removed the ‘primed’ kinase is fully active ,6. OtherV600E/K inhibitors vemurafenib and dabrafenib. These inhibitors were developed to selectively inhibit BRAFV600E/K and although they are effective in treating BRAFV600E/K-driven melanoma they cause adventitious tumour progression in non-melanoma tissue. This is because inhibitor binding to wild-type RAF isoforms induces RAS-GTP-dependent CRAF homodimers or BRAF-CRAF heterodimers in which the drug-bound protomer trans-activates the drug-free promoter leading to MEK1/2–ERK1/2 activation [The second and best-known example of paradoxical kinase activation is seen with the first generation BRAFtivation ,77 , 26 (XMD17-109) or AX15836 to the ERK5 kinase domain promotes NLS and TAD exposure which in turn promotes nuclear localisation and paradoxical activation of ERK5 transcriptional activity [We have recently shown that the binding of cpd activity . The ERK46 and BAY-885 in the MEF2D:GAL4 assay (25 (XMD17-26), cpd 35  and cpd 46 to ERK5 to ATP  and the αC helix, is not occupied by ATP. Comparison of the conformation of the ERK5 αC helix and activation loop in the presence and absence of inhibitors or ATP  so care must be taken when interpreting results. This highlights the need to find a selective paradox-breaking ERK5i or to use an alternative approach such as a PROTAC [These findings show that it is essential that all ERK5i are tested not just for off-target activity against kinases and bromo-domain containing proteins, but also for promotion of nuclear localisation and paradoxical transcriptional activation using appropriately sensitive assays. For ERK5 this can be assessed using the ERK5:MEF2D reporter assays, such as the GAL4-MEF2D driven GRE reporter . It shoua PROTAC  to complWe dedicate this article to the memory of Michael Wakelam a dear friend, mentor, colleague, a world leader in lipid signalling and lipidomics, and Director of the Babraham Institute until his death in the 2020 Covid-19 pandemic.Importance of the field: Protein kinase inhibitors can induce unintended paradoxical signalling. Paradoxical kinase activation can exacerbate disease, such as stimulating tumour growth. ERK5 nuclear localisation and transcriptional activity is paradoxically stimulated by ERK5i.Current thinking: Characterising the mode of action of kinase inhibitors on their targets using appropriately sensitive assays is critical for all drug discovery programmes. Determining whether ERK5i promote transcriptional activation of ERK5 is essential for any new potential ERK5 therapeutic.Future directions: Activation of ERK5 transcriptional activity by ERK5i raises significant questions about the role of ERK5 kinase and transcriptional activities in ERK5 biology, including disease biology and therapeutics.


The gap junction protein Connexin43 (Cx43) is highly regulated by phosphorylation at over a dozen sites by probably at least as many kinases. This Cx43 “kinome” plays an important role in gap junction assembly and turnover. We sought to gain a better understanding of the interrelationship of these phosphorylation events particularly related to src activation and Cx43 turnover. Using state-of-the-art live imaging methods, specific inhibitors and many phosphorylation-status specific antibodies, we found phospho-specific domains in gap junction plaques and show evidence that multiple pathways of disassembly exist and can be regulated at the cellular and subcellular level. We found Src activation promotes formation of connexisomes  in a process involving ERK-mediated phosphorylation of S279/282. Proteasome inhibition dramatically and rapidly restored gap junctions in the presence of Src and led to dramatic changes in the Cx43 phospho-profile including to increased Y247, Y265, S279/282, S365, and S373 phosphorylation. Lysosomal inhibition, on the other hand, nearly eliminated phosphorylation on Y247 and Y265 and reduced S368 and S373 while increasing S279/282 phosphorylation levels. We present a model of gap junction disassembly where multiple modes of disassembly are regulated by phosphorylation and can have differential effects on cellular signaling. Gap junctions are specialized membrane domains composed of tens to thousands of channels that permit intercellular exchange of ions and members of the metabolome of <1000 Da ,3,4,5  and saw that formation of connexisomes was associated with activated Src. We observed high levels of MAPK phosphorylation on S279/282 of Cx43 specifically on connexisomes in these cells. We show that Src mediated downregulation of gap junction size and number could be overcome by proteasome inhibition and led to 4.4-fold increase in pY247 levels while pY265, another src site, decreased, arguing that pY247 was specifically preserved within the gap junction. Thus, we propose that there are multiple means of gap junction turnover, and that kinase interactions with Cx43 can drive the method of internalization with phospho-specific subdomains playing a mechanistic role in this process.All general chemicals, unless otherwise noted, were purchased from ThermoFisher Scientific . ALLN, MG132, tetradeconoylphorbol acetate (TPA), cycloheximide (CHX), phenylmethylsulfonyl fluoride (PMSF), Brefeldin A (BFA), and a rabbit antibody against Cx43 (C6219) were from MilliporeSigma . Mouse anti-Cx43 antibodies, Cx43CT1 and Cx43IF1, were prepared against amino acids 360–382 of Cx43, antibody Cx43NT1 against amino acids 1–20 (Cx43NT1) of Cx43 , 2 environment. Plasmids containing Cx43-FPs were electroporated into cell lines via a Nucleofector apparatus . Stably transfected clones were isolated with cloning rings in the presence of the selective antibiotic hygromycin (200 µg/mL).Normal rat kidney (NRK) epithelial cells , LA-25 cells (NRK cells containing temperature sensitive v-Src ) and BWE3VO4, 2 mM PMSF, and 1x Complete protease inhibitors (MilliporeSigma) and cellular proteins were separated by SDS-PAGE (10% polyacrylamide). After electrophoresis, protein was transferred to nitrocellulose, the membrane was blocked, and incubated with antibodies to total and appropriate phospho-specific antibody overnight  as previously indicated )In the second video , we showThe phospho-profile of gap junctions compared to connexisomes, as shown in 4Cl increases ERK site phosphorylation while inhibiting src phosphorylation. On the other hand, inhibition of tyrosine phosphatases with vanadate increased the phosphorylation levels of Y247 and S279/282 in tandem. Whether this is due to direct effects on Cx43 or changes in src signaling is not clear at this time. Particular caution is necessary for interpreting protein degradation inhibitor data; proteasome inhibition has effects on many short-lived proteins that may be playing a role in regulating gap junction stability. Remarkably, we saw that proteasome inhibition could effectively counteract the effects of src activation on gap junction size and do so quite rapidly  homeostatic cells maintain stable gap junctions that engage in intercellular communication and serve as a scaffold for homeostatic signaling that can be rearranged through interaction with kinases in response to specific cues; (2) disaggregation and internalization of connexons into their parent cells downregulates both gap junction channel and scaffold function; (3) formation of connexisomes maintains scaffold function while separating it from channel function. This concept of differential scaffold functions and altered spatiotemporal regulation of these scaffolds by kinases adds to our understanding of how Cx43 can regulate cell behavior. Our finding that Src can actually hijack aspects of these processes offers insight into how post-translational modification of Cx43 may be targeted in a variety of pathologies to alter cellular signaling.


Our analytical and numerical approach is based on a Fokker–Planck equation for g(h) :148501, g(h) spreads during winter and contracts during summer. This behavior is in agreement with recent satellite observations from CryoSat-2 (Kwok and Cunningham in Philos Trans R Soc A 373(2045):20140157, g(h) is a probability density function, we quantify all of the key moments  as greenhouse-gas radiative forcing, much slower than do solely thermodynamic models. This exhibits the crucial role that ice mechanics plays in maintaining the ice cover, by redistributing thin ice to thick ice-far more rapidly than can thermal growth alone.We study the seasonal changes in the thickness distribution of Arctic sea ice,  The recent decline in both the areal extent and the average thickness of sea ice, as evidenced by satellite and submarine measurements, drives study of its origins . The keyh .To study the evolution of the ice volume, one could treat ice as a continuum and construct the mass, momentum, and energy balance equations . Howeverg(h). It is defined as follows: Consider a region with area R that is sufficiently large to contain a range ice of different thicknesses. Then the integralA / R) that contains ice of thicknesses between g(h) on space and time is implicit. The spatio-temporal evolution of g(h), subject to wind, thermal and mechanical forcing, is governed by [f is the thermal growth/melt rate of ice, and g(h)’s that were qualitatively similar to those from observations. However, Eq. The key step in the construction of such a description was taken in 1975 by Thorndike et al. , who interned by :2\documee et al.  separatee et al.  noted thg(h). In the first calculation he obtained g(h) was taken from observations. Depending on the values of Thorndike, in a later study , made twr (MU71) , and g(hd and g(h). The following is a brief summary of his findings:g(h) attains the maximum value at When g(h) on both sides of the maximum, but for Choosing In order to obtain a steady solution, it is necessary for g(h).The solutions with This study considerably improved our understanding of A closed form of entclass1pt{minimaIt was assumed that ice only from a particular range of thickness could ridge to produce thicker ice, but this is generally not the case .g(h).It was difficult to use this framework to study seasonal changes in The theoretical investigation of the evolution of g(h) was complemented by observations of thickness in the central Arctic, which revealed that r. Using dimensional arguments he related H to F / r. Thorndike [h. While this approach overcame limitation 2 from the previous study, the nonlinear integro-differential equation could only be solved numerically. The solutions displayed exponential tails, showing that the simple rules for the thermal and mechanical interactions were sufficient to obtain g(h) for thick ice that were in qualitative agreement with the observations.For his second calculation, Thorndike formulated the original equation as a Markov process; and by assuming the forms of horndike  thus conhorndike  argued tC is introduced to ensure that g(h) is normalized. Numerical solutions to Eq. Recently, Godlovitch et al.  generalirvations .Finally, the World Climate Research Programme Coupled Model Intercomparison Project Phase 5 (CMIP5) models, which use momentum equations for the ice pack and hence a wide range of constitutive models for the ice rheology, are known to poorly represent the spatial patterns of ice thickness . This hig(h). Observations indicate that a region with a length scale of 100 km or more is required to define g(h) [up to a few tens of meters in general [g(h). This line of reasoning led us to use an analogy with Brownian motion to interpret When studying Eq. ine g(h) , whereas general . Hence, cument}ψ . Now we The classical problem of Brownian motion concerns the motion of a pollen grain in water , 12. Thegh and from h to g. Here, g, are constants.We view the short length and time scales of individual mechanical processes (ridging and rafting) relative to the overall evolution of entclass1pt{minimaL as the horizontal length scale; R, Eq. g. In this paper, we discuss the analytical and numerical solutions to Eq. We nondimensionalize this equation by choosing S is Stefan number defined as q and H, this solution is unique.A unique steady solution to Eq. h yields Setting to zero the first derivative of Eq. tion Eq.  from ourh and t isAs a first step in understanding how the thickness distribution is driven by climatological forcing, we introduce a simple model for the growth rate, with mentclass2pt{minimT is the temperature of the upper surface. Ice export is S is large for ice, the energy balance across h is global and hence, for example, Seasonality and climate forcing are introduced by coupling Eq. m in Eq. , we haveh dependence of the albedo, where the characteristic length scale is the inverse of the spectrally averaged Beer’s extinction coefficient The typical growth rates from the EW09 model for winter and summer are shown in Fig. λ=0.67 m . This beX. Figure The mean thickness is defined as:entclass1pt{minimac models , 10. Impc models ), here wsee Fig. .Fig. 2Seg(h), we define ‘thin’ ice as ice of thickness For the purpose of discussing the changes in mum Fig. , roughlyg(h) at the middle and end of the growth and melt seasons respectively. For all g(h) to create more thick ice. As g(h) contracts towards thinner ice and the skewness increases, both of which are enhanced substantially as Figure ses Fig. .Fig. 3Seg(h) with the recent satellite observations from CryoSat-2 [g(h) during winter is explained as above within the framework of the theory; ice growth makes thicker the ice that is formed and subsequently deformed, shifting the peak to the right and broadening the distribution, a behavior that is suppressed as We make a qualitative comparison of ryoSat-2  in Fig. g(h) is known, all thickness dependent moments can be calculated. A quantity of keen interest is the albedo, whose summer mean values are difficult to model because of the concurrent presence of a wide range of ice thicknesses in the basin. We plot the seasonal evolution of the mean albedo Importantly, once  balance . Figuresg(h) can be understood by considering Eq. The effect of ons Fig. . Figure c models , 10.Fig.Finally, given the important shift in the transitions of the ice states  from solely thermodynamic models to the full mechanical and thermodynamic treatment of this theory, the response of the ice pack to a radiative flux perturbation is clearly different between these approaches. We have quantified the relaxation time scales for different initial conditions (see Appendix 3) and find that there is a range of thin ice fractions, g(h), of Thorndike et al. [f to understand the climatological evolution of g(h). In the simplest case, g(h) leads to the generation of multiple time scales, which in turn affect the evolution. Thus, as previously suggested by Thorndike [g(h) and its moments relax on different time scales, which clearly has important geophysical consequences.Using concepts and methods from statistical physics we have transformed the theory of the sea ice thickness distribution, e et al.  into a stion Eq. . The solhorndike , we do ig(h) from our model are in good agreement with the recent satellite measurements over the Arctic basin [g(h) spreads during the growth season and contracts during the melt season. As greenhouse gas forcing, A climatological suite of calculations was performed by coupling the Fokker–Planck equation to the thermodynamic model of Eisenman and Wettlaufer . The traic basin , 17. As ic basin , 10, we aneously  relative6 m Fig. .g(h) by decreasing the mean growth rate and the seasonally averaged mean thickness g(h) to reach a stationary state starting from an arbitrary initial condition range from The seasonal behavior of the thin-ice fraction g(h) due to Thorndike et al. [g(h) in climate models should lead to a more realistic representation of Arctic sea ice within them. The thermodynamic component used here [The results presented here demonstrate veracity of using the methods and concepts of statistical mechanics to study the geophysical-scale evolution of Arctic sea ice. As described in the introduction, the CMIP5 models poorly represent the spatial patterns of ice thickness . The cone et al.  was to ased here  reproducsed here , which i


Candida albicans, the small GTPase, Rsr1, guides the directional growth of hyphae that invade human tissue during life-threatening infections. Rsr1 is a Ras-like GTPase and a homolog of the conserved Rap1 subfamily, which directs migration in mammalian cells. Research into how this single GTPase delivers complex intracellular patterning is challenging established views of GTPase regulation, trafficking, and interaction. Here, we show that Rsr1 directly and indirectly coordinates the spatial and temporal development of key intracellular macrostructures, including septum formation and closure, vacuole dynamics, and nuclear division and segregation, as well as whole-cell morphology by determining branching patterns. Furthermore, we categorize these functions by differential Rsr1 localization and activity state and provide evidence to support the emerging view that the cytosolic pool of Ras-like GTPases is functionally active.Understanding how single eukaryotic cells self-organize to replicate and migrate is relevant to health and disease. In the fungal pathogen,  Candida albicans, are disrupted by deletion of the small GTPase, Rsr1, which localizes Cdc42 and its kinase, Cla4, to the site of polarized growth. We investigated additional abnormalities observed in rsr1Δ hyphae, including vacuole development, cytoplasm inheritance, mitochondrial morphology, septin ring organization, nuclear division and migration, and branching frequency, which together demonstrate a fundamental role for Rsr1 in cellular organization. Rsr1 contains a C-terminal CCAAX box, which putatively undergoes both reversible palmitoylation and farnesylation for entry into the secretory pathway. We expressed variants of Rsr1 with mutated C244 or C245, or which lacked GTPase activity (Rsr1K16N and Rsr1G12V), in the rsr1Δ background and compared the resulting phenotypes with those of mutants lacking Bud5 (Rsr1 GEF), Bud2 (Rsr1 GAP), or Cla4. Bud5 was required only for cell size and bud site selection in yeast, suggesting there are alternative activators for Rsr1 in hyphae. Septin ring and vacuole dynamics were restored by expression of unpalmitoylated Rsr1C244S, which localized to endomembranes, but not by cytoplasmic Rsr1C245A or GTP/GDP-locked Rsr1, suggesting Rsr1 functions at intracellular membranes in addition to the plasma membrane. Rsr1K16N or cytoplasmic Rsr1C245A restored normal nuclear division but not septin ring or vacuole dynamics. Rsr1-GDP therefore plays a specific role in suppressing START, which can be signaled from the cytosol. Via differential palmitoylation and activity states, Rsr1 operates at diverse cell sites to orchestrate proper nuclear division and inheritance during constitutive polarized growth. As cla4Δ phenocopied rsr1Δ, it is likely these functions involve Cdc42-Cla4 activity.Directional growth and tissue invasion by hyphae of the pathogenic fungus,  Candida albicans, an opportunistic fungal pathogen  at the hyphal tip  is tethered at the plasma membrane just behind the extending tip. As the tip extends, the septin ring accumulates regulators and effectors that organize septal closure after passage of a daughter nucleus into the apical cell  adjacent to the CAAX  box. This CCTIM motif suggests that Rsr1 C244 undergoes reversible palmitoylation, as occurs in C. albicans Ras1, while the terminal Met residue indicates farnesylation at C245  tag. Rsr1C244S and Rsr1C245A carried a mutation in the C-terminal Cys244 putative palmitoylation site and Cys245 CAAX box farnesylation site, respectively. The phenotypes of rsr1Δ mutants expressing Rsr1K16N or Rsr1G12V, which mimicked the GDP- or GTP-locked forms of Rsr1, respectively, were compared with those of strains deleted in Bud5 (Rsr1 GEF) or Bud2 (Rsr1 GAP). We identified five phenotypic groupings, each requiring differential regulation of Rsr1. All the phenotypes exhibited by rsr1Δ and hyperactive Rsr1 mutants were phenocopied by deletion of cla4. As Cla4 acts downstream of the Rsr1 effector, Cdc42, and upstream of cortical Rsr1 via Lte1 . Although no YFP-Rsr1C244S signal was observed at the PM, farnesylation may direct Rsr1C244S transiently to the PM via the normal trafficking pathway, as reported for S. cerevisiae Ras2 [ScRas2(SCaax)]  for proteolysis of AAX and methylation , 44. Palckground  to C. ThR for pro vacuole . Express(SCaax)] , where iytosolic . This isytosolic .10.1128/mBio.01666-20.1MOVIE S1Movie S1, MOV file, 0.6 MB.Rsr1 associates with the plasma membrane in a dynamic manner, with an intensity bias towards the hyphal tip. Time-lapse fluorescence microscopy of YFP-Rsr1 using spinning disk confocal microscope . Cells wCopyright © 2020 Bedekovic et al.2020Bedekovic et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the K16N (GDP-bound mimic) or YFP-Rsr1G12V (GTP-bound mimic) were imaged . YF. YFK16N al septa . The posrsr1Δ and bud2Δ mutants produce enlarged yeast cells with random budding patterns  was expressed in the rsr1Δ null background, and yeast cells were compared with rsr1Δ, bud2Δ, and bud5Δ mutants by staining with calcofluor white (CFW). All the mutants with altered Rsr1 GDP-GTP cycling and the cytoplasmic Rsr1C245A strain grew as enlarged yeast cells, and z-stack projections confirmed that bud sites were randomly positioned  or GDP-bound Rsr1 (Rsr1K16N or Rsr1C245A), irrespective of cytosolic or PM localization, were mononucleate. This indicates that Rsr1-GDP plays an isoform-specific role in the negative regulation of nuclear division.Yeast cells that are large and rounded are often multinucleate , 52. To nucleate . Deletiorsr1Δ mutant, RSR1 was deleted in a strain coexpressing YFP-tagged Nop1, a nucleolar protein, and Cdc3, a septin  that undergo synchronous nuclear division. Three daughter nuclei migrate through the presumptum (Cdc3-GFP) into the daughter compartment. Asynchronous division occurs in the mother compartment, but neither daughter migrates  migrating through the septin ring (Cdc3-GFP) prior to nuclear division followed by the retrograde migration of a daughter nucleus into the mother cell  without daughter nucleus segregation, and aberrant migration through the presumptum of either undivided nuclei or both daughter nuclei, followed by septal closure.rsr1Δ mutant, septin ring morphology, localization, and behavior were examined by imaging Cdc3-GFP or Cdc11-GFP in the rsr1Δ mutant and wild-type cells during hyphal growth . Inphal tip  and 5B. phal tip  and 5C. ry septa  and E. Try septa  to produbud5Δ mutant, the localization and morphology of septa were normal, with a thin, white, chitinous primary septum and the darker β-glucan layer of the secondary septum on either side  compared to images from the rsr1Δ strain (B). Presented is the medial focal plane. Download FIG S1, TIF file, 1.2 MB.Composite of septal phenotypes in DAY185 and Copyright © 2020 Bedekovic et al.2020Bedekovic et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 1–243, a truncated version of Rsr1 lacking the C-terminal membrane localization domain, was heterologously expressed in Escherichia coli and used in pulldown assays with C. albicans lysates from yeast and hyphal cells. Cdc42 GTPase and its GEF, Cdc24, were identified, as expected. The 5 core septins—Cdc3, Cdc11, Cdc12, Cdc10, and Sep7/Shs1—were detected at levels above that for the GST control in hyphae, although Cdc10 was not seen at a level greater than that of the negative control in yeast -6×His-Rsr1east see , confirmeast see .10.1128/mBio.01666-20.7FIG S21–243, lacking the C-terminal CAAX box, in yeast and hyphae and identified by mass spec. Data are presented as a ratio of signal against GST tag alone (background), n = 3. (B) Interaction of GST-Rsr1 with YFP-Bud5 and YFP-Bud2 was examined with a far-Western blot. After the treatment of the PVDF membrane with far-Western blot buffers, proteins were detected with anti-GFP antibody or incubated with purified GST-Rsr1 and detected with anti-GST antibody. Binding to the YFP tag alone was determined using a GFP control. Download FIG S2, TIF file, 0.8 MB.Rsr1 interacting partners. (A) Interacting partners were pulled down with heterologously expressed GST-Rsr1Copyright © 2020 Bedekovic et al.2020Bedekovic et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01666-20.8FIG S3n ≤ 26. Presented is the medial focal plane. Download FIG S3, TIF file, 0.8 MB.Spk stability in the hyphal tip. Aberrant sequential positioning of Mlc1-YFP during hyphal growth in PDMS chambers was visualized (A) and quantified (B). Aberrant Mlc1-YFP localization was defined as (i) nonlinear sequential positioning of Mlc1-YFP and (ii) loss of tip contact with the ridge, 15 ≤ Copyright © 2020 Bedekovic et al.2020Bedekovic et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the rsr1Δ mutant suggested that the signal coupling septal closure with nuclear segregation was compromised or delayed in this mutant. The septin collar is a scaffold for 83 septin-associated proteins that integrate multiple signals to coordinate downstream events in cytokinesis   . The rsrsr1C245A  and C. Hhenotype . Togethe1-phase compartments, mitochondria are small and peripherally localized until sufficient cytoplasm is synthesized to initiate START  is confined to older cells after sufficient uptake of nutrients for re-entry into the cell cycle, i.e., in cells that are furthest from the growing tip , and they emerged from the oldest cell  were formed within the same time period, primarily from the oldest cell but some from younger compartments. The same values were determined in the cla4Δ mutant. Thus, multiple distal compartments in these mutants initiated START compared to that in the control strain, a hallmark of loss of apical dominance, and confirmed that these cells retained metabolic activity. Interestingly, the total length of hyphae produced per mother yeast cell by these mutants was greater than the wild-type strain (see P ≤ 0.0001). Hence, there was no net increase in overall growth, as increased hyphal extension in one subpopulation of cells was countered by a high rate of bursting and loss of viability in another  Net hyphal growth at the population level in WT, Rsr1 reintegrant, and hyperbranching mutants after cell lysis. (C) Aberrant septum localization and cell morphology of cla4Δ yeast. Yeast cells were grown for 16 h in YPD at 30°C and stained with CFW. Aberrant septa are marked with closed arrows. Download FIG S4, TIF file, 0.6 MB.Morphological changes at the whole-cell level. Rsr1 dysregulation resulted in longer hyphae. (A) Total hyphal length, determined as a sum of primary hyphal filament and lateral branches, was measured after growth in 20% FCS for 5 h. For Copyright © 2020 Bedekovic et al.2020Bedekovic et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the G12V, consistent with the findings for the bud2Δ mutant, but was instead rescued by expression of Rsr1K16N, Rsr1C244S, or Rsr1C245A. Expression of Rsr1C244S had already been shown to restore normal vacuole morphology, cytoplasm distribution, and septin ring organization, which are prerequisites for supporting apical dominance. However, expression of GDP-locked Rsr1K16N or cytoplasmic Rsr1C245A was specifically able to restore apical dominance, even though the cells retained fragmented vacuoles and increased cytoplasm. This strongly suggests that the Rsr1-GDP isoform acts as a negative regulator of START.Loss of apical dominance was not rescued by expression of Rsr1C. albicans -Rsr1-Bud2(GAP) signaling module (Group 1 consisted of a single g module  and Cla4bud5Δ null (see below “The GEF Bud5 is largely dispensable for Rsr1 function”). Although the group 2 phenotype was Bud5 dependent, it was rescued in the rsr1Δ mutant by expression of unpalmitoylated Rsr1C244S. All other components of the Rsr1 signaling module were required for the wild-type morphology, implying that, although visualized only at endomembranes, Rsr1C244S must be able to under GDP-GTP cycling. As Bud5-GFP resided at the cell periphery and Bud5 was required for normal yeast cell morphology, it is plausible that farnesylated Rsr1C244S was trafficked to the PM where it was activated. The similarly modified Ras1C288/7S isoform was reported at the cell periphery in C. albicans, indicating that this trafficking route is possible in farnesylated but unpalmitoylated GTPases , including septin ring organization. Septin mutants form unstable septin rings, are multinucleate, and are defective for invasive growth  Rsr1, Bud2, GDP-GTP cycling, and the Cla4 kinase . It was e growth . Rsr1C24C245A exhibited the rsr1Δ null phenotype. This group was defined by Bud5 independence and rescue by expression of Rsr1C244S  requires association with cytoplasmic chaperones , which sequester the hydrophobic acyl group  and those that additionally require palmitoylation for more stable PM association and/or raft/domain targeting (bud site selection and substrate penetration)  . None ofK16N or Rsr1C245A  medium  at 37°C.The Plasmids and primers are listed in RSR1.(i) Site-directed mutagenesis of  Vector pMG2128 (RSR1 open reading frame (ORF) CR_02140W_A 825 bp upstream of the promoter and 240 bp downstream of the stop codon, was modified by site-directed mutagenesis (SDM) using primer pairs 1-2, 3-4, and 5-6, complementary to the RSR1 ORF. Primer silent mutations introduced BamHI (C244S), NarI (K16N), and ScaI (G12V) cut sites for screening. Resulting plasmids pAB146 (Rsr1G12V), pAB147 (Rsr1K16N), and pAB144 (Rsr1C244S) were sequenced before linearization with MluI and integrated into the promoter of the disrupted rsr1 locus in CA8832 , generating strains A498, A500, and A494. Cells were selected for uridine prototrophy and confirmed by PCR. pMG2128 , containRSR1.(ii) FP-tagging and SDM of YFP- The codon-optimized YFP sequence with a Start codon  with a 6-amino-acid (aa) linker (CCGCCATGGCTCGAGGGT). The fragment encoding YFP-RSR1 and flanked by MluI (5′) and SalI (3′) sites was synthesized by GeneArt (Life Technologies) and inserted between MluI and SalI sites in the pACT1 plasmid , pAB209 (YFP-Rsr1K16N), pAB210 (YFP-Rsr1C244S), and pAB213 (YFP-Rsr1C245A), respectively. Plasmid DNA was linearized with StuI and integrated into the RPS1 locus. Transformants were confirmed by PCR, generating strains A582, A572, A574, A577, and A578. Plasmid pACT1-mtGFP bearing a mitochondrial localization signal  PCR-mediated C-terminal FP tagging: Cdc11-GFP, Mlc1-YFP, and Dbf2-GFP. Cdc11 (C5_00070W_A), Mlc1 (CR_03090C_A), and Dbf2 (C2_06670C_A) were C-terminally tagged by amplifying the FP-nourseothricin (NAT) cassette from plasmids pGFP-NAT1 or pYFP-NAT1 (YFP-NAT1  using prRSR1 ORF in Nop1-YFP Cdc3-GFP background.(iv) CRISPR-Cas9 to disrupt  The CRISPR-Cas9 system for C. albicans (RSR1 ORF (CR_02140W_A). A unique guide sequence (AATCACCGTGCAATTTGTCC) targeting both alleles 142 nt from the RSR1 start codon (located outside the intron from 8 to 81 nt) was cloned into plasmid pV1200. A mutagenic repair DNA template (primer pair 27-28) containing a stop codon, a cut site, and a frameshift was amplified, and pV1200 containing RSR1 PAM-142 (primer pair 25-26) was cut with KpnI and SacI and cotransformed into 8860 (Nop1-YFP Cdc3-GFP), generating strain A827 (rsr1Δ/Δ Nop1-YFP Cdc3-GFP). Cells were selected on YPD agar with 300 μg/ml nourseothricin. The NAT resistance marker was recycled, and the RSR1 ORF was sequenced.albicans  was used1–243.(v) Heterologous expression of GST-Rsr1RSR1 was amplified from plasmid pAB158 using primer 17  and 18 , digested with ApaI and XhoI, and ligated into pET41b-FT, a derivate of commercial plasmid pET41b (Invitrogen) (E. coli BL21(DE3).itrogen) , to geneitrogen) . A GST-o(i) Mlc1-YFP imaging in PDMS chambers. Polydimethylsiloxane (PDMS)  chambers were used for imaging Mlc1-YFP as described by Thomson et al.  oil immersion lens objective (Carl Zeiss) and a 16-bit CoolSNAP H2 charge-coupled-device (CCD) camera (Photometrics) with attached incubation chamber (PeCon GmbH) at 37°C. YFP was imaged using a YFP-2427B-ZHE-ZERO filter (Laser 2000). Spk positioning of Mlc1-YFP was plotted in hyphae for (i) nonlinear sequential positioning of Mlc1-YFP and (ii) loss of ridge contact. Septal closure duration was determined where t = 0 represents the time of appearance of Mlc1-YFP at the septum  YFP-Rsr1, YFP-Rsr1 Hyphae were grown on a poly-l-lysine slide for 5 h. Images were acquired as described, with 2 × 2 binning and a 3-s exposure. YFP was imaged as before. Images were deconvolved in AxioVision (Rel. 4.8) software, and YFP intensity was measured at the plasma membrane along the 10-μm region subapical to the hyphal tip using ImageJ (v. 1.45s).(iii) FM4-64 staining and analysis of vacuolar area. Vacuoles were stained with the lipophilic styryl red dye FM4-64 . Hyphae were grown on a poly-l-lysine slide for 5 h and stained with 16 μM FM4-64 for 45 min at 37°C in the dark, washed with double-distilled water (ddH2O), and incubated in fresh 20% FBS for 1 h at 37°C. Samples were co-stained with 5 μg/ml calcofluor white (CFW) (Sigma), and 0.4-μm optical sections were captured on an UltraVIEW VoX spinning disk confocal microscope  using 561-, 514-, and 405-nm lasers and running Volocity 6.3 software (PerkinElmer). Vacuolar area (μm2) was determined by tracing vacuoles in a two-dimensional (2D) format of a maximum projection of optical sections using ImageJ (v. 1.52p). Total cell volume was measured by tracing CFW-stained nonbranched subapical compartments. The proportion of cytoplasm per hyphal compartment was calculated by subtracting vacuolar area from total cell area.(iv) Live-cell imaging of FP-tagged proteins. Hyphae were grown in an 8-well ibidi μ-slide (Ibidi) and imaged every 3 min, or as indicated, on a spinning disk confocal microscope. Optical sections were acquired, and images are maximum intensity projections, unless otherwise stated. Where indicated, the growth medium was supplemented with 2 μg/ml CFW. For mitoGFP, hyphae were incubated for 5 h at 37°C and stained with 5 μg/ml CFW. Optical sections at 0.4-μm increments were captured on a spinning disk confocal microscope, using 488-and 405-nm lasers. The mean fluorescence intensity was analyzed for 5-μm2 areas in the middle of the nonbranched subapical hyphal compartments in ImageJ (v. 1.52p).(v) Quantification of nuclei and cell diameter. Hyphae were grown on a poly-l-lysine slide for 5 h and fixed in 4% formaldehyde. Nuclei were stained with 1.2 mM DAPI (Sigma) or with Vectashield with 1.5 mg/ml DAPI (Vector Laboratories). Hyphae were co-stained with 0.1 μg/ml CFW where indicated. Optical sections were captured on the Zeiss Axio Observer (40×/1.3 NA), using a DAPI filter. Nondividing yeast cells were analyzed for nuclear number and cell diameter.(vi) CFW staining of bud scars. Hyphae were grown as before and stained with 10 μg/ml CFW. Bud scars were visualized after 16 h of incubation at 30°C. Yeast cells were washed with ddH2O and stained with 15 μg/ml CFW. Z-stacks were captured on a Zeiss LSM880 with Airyscan running Zen 2.3 SP1, with maximum intensity projections generated in Zen.(vii) Cell lysis and secondary growth axis analysis Cell lysis was determined with live imaging of cells incubated in either YPD at 30°C or 20% FBS with 2% glucose and with or without 1 M sorbitol at 37°C in an 8-well ibidi μ-slide (ibidi). Images were acquired every 5 min using a differential interference contrast (DIC) filter. Cell lysis was quantified for each frame until no new cell death was detected (≈1 h). The total number of axes per primary hypha was determined after 5 h of incubation.E. coli BL21(DE3) bacterial cultures were diluted to an optical density at 600 nm (OD600) of 0.1 in 1 L LB broth supplemented with 25 μg/ml kanamycin and grown to an OD600 of 0.8. Expression was induced in 1 mM IPTG , and cultures were incubated overnight at 30°C, 200 rpm. Cells were lysed with low-salt buffer  and freezing, followed by incubation with 80 mg lysozyme for 1 h at 4°C and sonication. Cell lysate were ultracentrifuged , and the supernatant was retained. Protein extracts containing GST-Rsr1 were applied to glutathione columns  previously equilibrated in GST buffer . Columns were washed with 20 mM Tris and 500 mM NaCl (pH 7.4), and proteins were eluted with GST basis buffer containing 20 mM glutathione.Overnight 600 of 0.05 to an OD600 of 0.8 in YPD at 30°C and 200 rpm, for hyphae, overnight cultures were diluted to an OD600 0.05 in 20% FBS and incubated at 37°C and 200 rpm to an OD600 of 0.35. Cells were lysed with acid-washed glass beads in lysis buffer . Purified recombinant GST-Rsr1 (550 μg) was combined with 45 mg crude protein extract from C. albicans yeast or hyphae. Samples were incubated overnight on a rotor at 4°C. Glutathione Sepharose beads (500 μl) were equilibrated in phosphate-buffered saline (PBS), added to protein mixtures, and rotated for 3 h at 4°C. Beads were pelleted and washed with PBS. Proteins were eluted in 100 μl PBS with 20 mM glutathione and digested for 8 h at 37°C on an Investigator ProGest robotic workstation . Peptide solutions were dried in a concentrator , dissolved in 10 μl 0.1% formic acid, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using an QExactive Orbitrap mass spectrometer (Thermo Fisher Scientific). Mass lists were input to Mascot MS/MS ion searches  against sequence files from Candida Genome Database. Area values were calculated from the ion intensities of unique peptides identified for each protein. Extracted ion chromatograms for the 3 most abundant peptides were reconstructed in Mascot, and the area under the peak was calculated. Area values of proteins copurified with Rsr1 were normalized per sample using a ratio over the sum area value. Normalized values of individual proteins were divided by their normalized values in the negative control (nonspecific binding to the GST tag).After growth from an OD2 for 3 h at room temperature (RT). GST-Rsr1 was detected with horseradish peroxidase (HRP)-conjugated anti-GST antibody  using a chemiluminescence two-component kit . The membrane was stripped using stripping buffer (Thermo Fisher Scientific) for 15 min at RT, and YFP-labeled proteins were detected by anti-GFP antibody , followed by HRP-conjugated anti-mouse antibody .The protocol was according to that described by Wu at al. . BrieflyA homology model of Rsr1 (ORF CR_02140W_A) was generated using Yasara software (YASARA Structure 11.12.31) applying the following parameters: PsiBLASTs-1, EValue Max-0.5, Templates Total-5, Templates SameSeq-1, OligoState-4, Alignments-5, LoopSamples-50, TermExtensions-90, Speed-Slow. The core domain was modeled with a structural template identified by BLAST search in the Protein Data Bank (PDB) for a match, with a modeling E value cutoff of 0.5. Since the C terminus of Rsr1 is unique and does not show any homology to known protein structures, it was modeled by the YASARA algorithm for termini building.4 in acetone) with a Leica EMAFS2 and embedded in Spurr resin (Sigma). Additional infiltration was provided under a vacuum at 60°C before embedding in Leica FSP specimen containers and polymerized at 60°C for 48 h. Sections (60 nm) were prepared with a Diatome diamond knife on a Leica UC6 ultramicrotome and stained with uranyl acetate and lead citrate for examination with a JEOL 1400 plus transmission microscope  and imaging with an AMT UltraVUE camera (Deben).Cells were fixed by high-pressure freezing with an EMPACT2 high-pressure freezer and rapid transport system (Leica Microsystems Ltd.). Cells were freeze-substituted in substitution reagent (1% [wt/vol] OsO4 cells/5-μl spot on Spider agar (Cells were diluted to 2.5 × 10der agar . Colony Data generated in this study were imported into GraphPad Prism V8 for Windows (GraphPad Software) or SPSS V26. The statistical tests used, level of significance, and error bars are described in figure legends.